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Objective To investigate the pathway of lymphatic drainage of proteins from cerebral parenchyma in subarachnoid hemorrhage rat models. Methods Healthy adult male Wistar rats were divided into Saline group, Evans blue-labeled albumin (EBA) group, and SAH + EBA group. SAH models were produced by double injection of autologous arterial blood into cisterna magna. Using a modified microinjection method, EBA was injected into left candate-putamen of the EBA group and EBA + SAH group rats. In Saline control group, saline was injected. After injection, at 12 hours, 1 day, 2 days, 3 days and 5 days, the animals were sacrificed and the fluorescence signals of EBA were imagined and analyzed along the possible lymphatic drainage pathway, e.g. the brain tissue, the wall of common carotid artery, and cervical lymphatic nodes. Results One day after injection, in EBA group, the fluorescence of EBA initially appeared on the left of the brain, the wall of common carotid artery, left lateral cerebral ventricle, and the perivascular spaces of cerebral vessels. The fluorescence signals gradually expanded to the opposite side.Large amount of fluorescence granules accumulated in the outer layer of common carotid artery. Fluorescence was also found in cervical lymphatic nodes. Two days after injection in this group, the density of fluorescencein the brain became weaker while the density of fluorescence in rhinencephalon became stronger. The fluorescence of EBA was found in lymphatic nodes adjacent to abdominal aorta. In SAH + EBA group,reduced amount and velocity of the drainage of EBA from left caudate-putamen to rhinencephalon, cervical lymphatic nodes, and lymphatic nodes adjacent to abdominal aorta were observed. From 12 hours to 5 days after injection, fluorescence intensity of EBA in deep cervical lymphatic nodes in SAH + EBA group(8.9 ±2. 0, 11.9 ± 2. 5, 17.4 ± 3.7, 26.7 ± 4. 5 and 59.0 ± 8. 1 ) were lower than those in EBA group ( 14. 5 ±3.2, 27.5 ±7.4, 60.3 ±12.3, 138.0±12.0 and 108. 1 ±13.4, F=13. 17, 24.04, 66.81, 302.77 and 59.36, P ＜ 0. 01 ). From 2 to 5 days, fluorescence intensity of EBA in lymphatic nodes adjacent to abdominal aorta was also lower in SAH + EBA group( 11.0 ± 1.5, 12. 5 ±2. 8, 23.6 ±3. 2) than those in EBA group(26. 3 ±5.9, 47.5 ±9.6, 41.0 ±9.3; F =38. 17, 72.52, 19.01, P ＜0.01). Conclusion SAH can result in reduced drainage of macromolecular substances, e.g. protein, from the brain via lymphatic pathway.
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Aim To establish a method to evaluate lymphatic drainage of macromolecular tracer in cerebrospinal fluid in rats.Methods Rat cervical lymphatic blockade(CLB)models were established by occlusion of cervical lymphatic tubes and removal of cervical lymphatic nodes.The rats were divided into non CLB(normal controls) and CLB groups.~(125)I-labeled human serum albumin(~(125)I-HSA)was injected into the left lateral cerebral ventricle,and blood samples were collected and ~(125)I-HSA concentrations were detected continually within 24 hours.Concentration-time curve was drawn according to the single compartment model in pharmacokinetics.Parameters of pharmacokinetics such as area under curve(AUC),maximum concentration(C_(max)),transfer rate constant K_a and peak time(T_(max))were derived.The AUC,C_(max),K_a,and T_(max) regarding the lymphatic drainage of ~(125)I-HSA were calculated based on the differences between the two groups.Results AUC,C_(max),K_a of ~(125)I-HSA by lymphatic drainage were 51.97 mg·L~(-1)·h~(-1),2.91 mg·L~(-1),and 0.64 h~(-1),respectively.The proportion of AUC,C_(max),K_a of ~(125)I-HSA by lymphatic drainage to those of drained by both arachnoid granulations and lymphatics was 71.53%,44.02%,58.18%,respectively.T_(max) in CLB group(8.36±0.82 h)was much longer than that in non CLB group(3.57±0.54 h).Conclusions A method to evaluate lymphatic drainage of macromolecular tracer in cerebrospinal fluid in rats is successfully established.The lymphatic drainage pathway plays an important role in eliminating macromolecular substances in cerebrospinal fluid.
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Objective To observe the change of diffusion upper limit of macromolecules through pathological retina and the difference between the layers of retina. Methods Retinal edema was emulated by establishing branch retinal vein occlusion(RVO)model in miniature pig eyes under photodynamic method.Two days later,the retinas of both eyeballs were peeled off.The diffusion test apparatus was designed by ourselves.FITC-dextrans of various molecular weights(4.4,9.3,19.6,38.9,71.2 and 150 kDa)and Carboxyfluorescein(376 Da)were dissolved in RPMI-1640 solutions and diffused through inner or outer surface of retina.The rate of transretinal diffusion was determined with a spectrophotometer.Theoretical maximum size of molecule(MSM)was calculated by extrapolating the trend-linear relationship with the diffusion rate.In separate experiments to determine the sites of barrier tO diffusion,FITC-dextrans were applied to either the inner or outer retinal surface,processed as frozen sections.and viewed with a fluorescence microscope. Results FITC-dextrans applying tO inner retinal surface,4.4 kDa dextrans were largely blocked by inner nuclear layer(INL);19.6-71.2 kDa dextrans were blocked by the nerve fiber layer(NFL)and inner plexiform layer;150 kDa dextrans were blocked by NFL.FITC-dextrans applying to outer retinal surface,most dextrans with various molecular weights were blocked before outer nuclear layer(ONL).No matter applying to the inner or outer surface,Carboxyfluorescein can diffuse through the whole retina and aggregate at INL and ONL.After RVO,the inner part of retina became edema and cystoid,loosing the barrier function.Compared with the normal retina,the MSM in RVO tissues increased(6.5±0.39nm Vs 6.18±0.54nm,t=4.143,P=0.0001). Conclusions After RVO,the barrier function of inner part of retinal is destroyed and the upper limit of diffusion macromolecule size increased.which is nevertheless limited.ONL acts as bottle-neck barriers to diffusion,if the outer part of retina is damaged,the change of the diffusion upper limit will be prominent.