| BACKGROUND:In spite of the fact that there is a drastic drop in rebleeding due to the progress of the surgical treatment of cerebral aneurism, the prognosis of subarachnoid hemorrhage (SAH) does not improve because of a concurrent increase in the occurrence of secondary cerebral ischemia. It has been shown that secondary cerebral ischemia is one of the main complications of SAH and contributes to the high incidence of mortality and disability from this disease. Thus, understanding of the pathogenesis, and searching for a valid prevention and cure measure of secondary cerebral ischemia is of great importance in improving the prognosis of patients with SAH. The main cerebral arterial spasm, namely cerebral vasospasm (CVS), can not completely explain the secondary cerebral ischemia and related neural functional lesion and handicap. In fact, the sharp increase of intracranial pressure and the dysfunction of cerebral microcirculation after bleeding should not be ignored. Due to the damage of blood-brain barrier and cells etc. after cerebral ischemia secondary to SAH, a large amount of proteins and macromolecules are accumulated in the brain and further aggravate brain injury. The lymphatic drainage pathway that may play an important role in the clearance of macromolecules in the brain has not been paid so much attention as it should be. It has been found that hypoxia could provoke the expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhension molecule-1 (PECAM-1), and promote the compensatory angiogenesis in the brain after cerebral ischemia. Both clinical researches and animal experiments have confirmed that a large dose of pyridoxol could play a therapeutical effect on lymphostatic encephaledema, nevertheless the related mechanisms of action is not clear.OBJECTIVES:1. To investigate the changes of lymphatic drainage pathway of macromolecules from the brain substance of rats after subarachnoid hemorrhage (SAH) and the influence of pyridoxol on this pathway.2. To determine the effects of SAH on lymphatic drainage pathway of macromolecules from the cerebrospinal fluid (CSF) of rats and the influence of pyridoxol.3. From the cerebral angiogenesis view, to investigate the effects of cervical lymphatic blockade (CLB) on cerebral ischemic injury secondary to SAH and the influence of pyridoxol.METHODS:1. In this study, adult male Wistar rats were used as experimental animals. Cisterna magna injection twice of freshly autologous arterial blood was used to induce SAH in rats, and CLB model in rats was established by occlusion of cervical lymphatic tubes and removal of cervical lymphatic nodes. The rats were divided into NS (normal saline), control, SAH, SAH+CLB and pyridoxol groups. Evans blue-labeled albumin (EBA) was injected into the left caudato-putamen of the rats with improved microinjection method and fluorescence protein tracer technic. Rats were sacrificed at the 0.5, 1, 2, 3, 5 days after injection, and the distribution of EBA in the brain, subarachniod space (SAS), olfactory bulbs, common carotid arteries, cervical lymph nodes and abdominal paraaortic lymph nodes were observed and compared by macroscopy and fluorescence microscope. The same dose of normal saline was injected into the NS group rats to observe if the tissues above-mentioned had spontaneous fluorescence.2. CLB models in rats were established by occlusion of cervical lymphatic tubes and removal of cervical lymphatic nodes, and cisterna magna injection twice of freshly autologous arterial blood was used to induce SAH in rats. The rats were divided into control (including non-CLB and CLB), SAH (including SAH-non-CLB and SAH+CLB) and pyridoxol groups. 100μg of 125I-labeled human serum albumin (125I-HSA; CSF tracer) was injected into the left lateral cerebral ventricle. Arterial blood was sampled for 24h, and the concentration of CSF tracer recovery in plasma was monitored. According to the plasma tracer concentration vs. time curve, we calculated several pharmacokinetics parameters [such as AUC (area under concentration-time curve), Ka (transfer rate constant), etc.] to evaluate and compare a relative amount and transfer rate of the tracer transferred from the CSF to the plasma via lymphatic drainage pathway in each group.3. Cisterna magna injection twice of freshly autologous arterial blood was used to induce SAH in rats, and CLB model in rats was established by occlusion of cervical lymphatic tubes and removal of cervical lymphatic nodes. The rats were divided into control, SAH, SAH+CLB and pyridoxol groups. The behavior changes of rats after SAH were observed. Rats were sacrificed at the third day after SAH except control group sacrificed directly. Took the brain of rats and made them into the coronal frozen sections. The expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhension molecule-1 (PECAM-1) in the brain were determined by immunohistochemistry respectively. The density of angiogenesis in the brain was counted under fluorescence microscope.RESULTS:1. Control group at the 1d, SAH group at the 3d, SAH+CLB group at the 5d and pyridoxol group at the 3d after EBA injection, the fluorescent signals distributed intensively in the left caudato-putamen, diffusely in the left corpus callosum and reached to the contralateral hemisphere with various extents in different groups. Choroid plexus of lateral ventricles and periependyma had fluorescence staining, but it was stronger in the left lateral. Fluorescence spread into the perivascular space (PVS) in the left caudato-putamen, cerebral cortex and SAS. Fluorescent signals were found in the wall of the common carotid arteries, and showing red waved elastic membranes in the tunica media and red punctiform granules in the adventitia. The cervical and abdominal paraaortic lymph nodes were dotted with red fluorescent signals and the fluorescence mainly distributed along cortical and medullary lymph sinuses. The fluorescence in the cervical lymph nodes seemed stronger than in the abdominal paraaortic lymph nodes. Compared with control group, the drainage of EBA from the brain to the olfactory bulbs, cervical lymph nodes and abdominal paraaortic lymph nodes reduced after SAH. Compared with SAH+CLB group, the drainage of EBA from the brain to the olfactory bulbs and abdominal paraaortic lymph nodes were increased with pyridoxol.2. AUC and Ka of control group transfered via lymphatic drainage pathway took (%transferred via arachniod villi and lymphatic drainage pathways) 71.53% and 58.18%, respectively; AUC and Ka of SAH group transfered via lymphatic drainage pathway took 59.97% and 46.15%, respectively. Compared with control group, AUC and Ka of SAH group transfered via lymphatic drainage pathway decreased 60.59% and 43.75%, separately. AUC and Ka of SAH+CLB+pyridoxol group were 2.09 and 1.60 times separately as many as SAH+CLB group.3. Besides control group, rats of other groups appeared a series of abnomal behavior changes after SAH. Above all, SAH+CLB group was the most serious, and pyridoxol group improved obviously. The expression of PECAM-1 and VEGF in the rat brain of SAH group were significantly more than control group, SAH+CLB group increased further more than SAH group, but pyridoxol group decreased obviously compared with SAH+CLB group.CONCLUSIONS:1. Macromolecules in the brain could be drained via lymphatic pathway mainly to cervical lymph nodes, and part to abdominal paraaortic lymph nodes. SAH may induce a relative functional inadequacy of the lymphatic drainage pathway of macromolecules in the brain. Pyridoxol may improve the drainage of macromolecules in the brain via lymphatic pathway to some extent.2. Macromolecules in the CSF of both nomal and SAH rats are cleared mainly via lymphatic drainage pathway. Clearance of macromolecules in the CSF via lymphatic drainage pathway reduce after SAH, but it may be improved by using pyridoxol.3. CLB could aggravate the cerebral ischemic injury secondary to SAH, but pyridoxol may relieve the aggravation of the cerebral ischemic injury by CLB after SAH.
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