| BACKGROUD:Emergent cerebrovascular dissease is one of the three death causes in the world. Among the total, the incidence of subarachnoid hemorrhage is second only than brain infarction and cerebral hemorrhage. Majority of SAH is caused by brain rupture of aneurysm. Case fatality is above of 25%, about of 1/3 survivals's existence depend on other because of neurologic impairment, which bring albatross to family and society rehaemorrhagia and secondary cerebral ischema are the main complications of SAH and contributes to the mal-prognosis. In spite of the fact that there is a drastic drop in rebleeding due to the progress of the surgical management of cerebral aneurism, the outcome of SAH dose not improve because of a concurrent increase in the occurrence of secondary cerebral ischemia. Thus, understanding of the pathogenesis, preventionand management of secondary cerebral ischemia is of great importance in improving the outcome of patients with SAH.Over the past several decades, the main cerebral arteril spasm, namely cerebral vasospasn(CVS), has become a focus. In recent years, scientists discovered that the time course and severity of CVS are not completely parallel relation with secondary cerebral ischemia. In spite of CVS, the factor (such as cerebral micrangium vasospasm and abnormal regulate function of microcirculation etc.) are concerned with development of secondary cerebral ischemia. Neuronic death include apoptosis and necrosis. Mammalian cell apoptosis is regulated by Bcl-2 and caspase proteins, apoptosis protein activating factor(Apaf-1). The member of caspase family is belong to different apoptosis pathway, of the total, caspase-3 closely related to Apoptosis. In regulation of mitochondria in apoptosis, the protein of Bcl-2 family can regulate, the liberation of some apoptosis factor, thus it's important in determination on life-and-death of cell.It has been proved that macromolecular substance in brain and subarachnoid space could be drainage into extracranial lymphatic vessels and lymph nodes, by technique of macromolecular substance tracing in any kind of animals including of human. Pyridoxol is chemical compound of B vitamins, and important coenzyme in vivo. It participates in metabolism of amino acid and lipids and immune response. In clinic, the symptoms of the lymphostatic encephalopathy(LE) patients obviously relieved by using of high-dose Pyridoxol or bepanthen, which explain that pyridoxol can improve drainage of macromolecular substance. But the mechanism is unclear. After occurrence of secondary cerebral ischema following SAH, mass macromolecular substance (such as plasma protein etc.) can pass through the impaired blood-brain barrier into brain tissue.Because of damaged cell or ischemia metabolism fall, mass cell cleaved product and peptides are producd and rapidly increased in brain tissue. Accumulation of above macromolecular substance can damage directly, or induce the brain edema by osmotic pressure of brain tissue rising, even cerebral hernia, which lead to neurologic impairment or die. It's important to final termination that how the macromolecular substance are pellated. Nevertheless, other scholars have not refered to the inportant problem of macromolecular substance cleaning on secondary cerebral ischema following SAH at home and abroad at present.OBJECTIVE:Firstly, to investigate the important role of the cerebral lymphatic drainage pathway on the drainage of macromolecular substance and homeostatic equilibrium in brain tissue under pathological conditions.Sencondly, to determine the variability features of the cerebral lymphatic drainage pathway, and to investigate the endogenous influence of cerebral lymphatic drainage pathway on the development of secondary cerebral ischema following SAH.Thirdly, to search for the drug to prevent secondary cerebral ischema following SAH effectively, by improving cerebral lymphatic drainage of macromolecular substance in brain.MOTHODS:1. The healthy Wister rats were randomly divided into five groups:normal control, SAH group, SAH+CLB group, SAH+CLB+ pyridoxo group, SAH+CLB+ normal sodium group. Rat SAH model was established by the cisteme magna double injection of autologus arterial hemolysates. Rat cervical lymphatic blocked(CLB) model was established by occlusion of cervical lymphatic tubes and removal of lymphatic nodes. Dynamic alteration of physiological parameters, intracranial pressure(ICP), and pial microcirculation were determined within 12 hours. In vivo BA and responsiveness of BA to Ach measurement was performed after 48 hours.2.48 hours after induction, changes of the structure and form of cerebral cortex and hippocampal were detected by haematine eosin stain and PI stain. Apoptosis and cell injury in the cerebral cortex and hippocampal were detected by TUNEL method. The protein expressions of Bcl-2 and caspase-3 were assessed by SABC immmunohistochemistry. The expression of mRNA was assessed by technic of RT-PCR.RESULT:1. The physiological parameters remained within normal range with the exception of a temporary increase in arterial blood pressure(BP). Rats in SAH, SAH+CLB and SAH+CLB+normal sodium groups showed depressed EEG, increased ICP and a drop in cerebral perfusion pressure(CCP) arid rCBF. BA diameters and BA responsiveness to Ach were decreased. Disturbances of pial microcirculation were obviously abnormal, blood flow much presented flow of sediment, even could be found blood flow stasis and movement. Among the total, Rats in SAH+CLB and SAH+CLB+normal sodium groups were more serious. Pyridoxol may relieve the symptom mentioned above of CLB after SAH.2. (1) By HE stain, the loss of neurons, karyopyknosis in partial neurons and anachromasis were found in SAH group. In SAH+CLB group, the edema of brain tisssue and capillary wall, wider tissue space, edema of vessel wall was found. The number of neurons decreased and the arrangement were out of order. The nuclei were deep stained and in shape of triangle, strip or abnormity, and fartly found nuclear fragmentation. Pyridoxol may relieve the above symptome of CLB after SAH.(2) By PI stain, some neurons showed apoptotic conformation. Cell nucleus were in shape of ripple or crease, part of caryotins were pyknotic, individual caryotin were high agglutinated, marginalizated, and in crescent-shaped in SAH group. A great of neurons showed apoptotic conformation, and belong to advanced stage:caryotin were high agglutinated, marginalizated, and in crescent-shaped, and found nuclear fragmentation and apoptotic body in SAH+CLB group. Apoptosis were obviously decreased in pyridoxol group.(3) Scattered apoptotic cells were observed in SAH group, and a devil of apoptotic cells were observed in SAH+CLB group. Apoptotic cells were centralized in cerebral cortex, hippocamp, basal ganglia, choroid plexus and endyma etc. Of the total, hippocamp, endyma were more visible. In pyridoxol group, scattered apoptotic cells were observed.(4) By immunofluorescence technic, some expression of caspase-3 and Bcl-2 were observed in SAH group.In SAH+CLB group, the expression of caspase-3 was more than SAH group. However, the expression of Bcl-2 was less. In pyridoxol group, the result of expression of caspase-3 and Bcl-2 were opposite to SAH+CLB group.(5) By RT-PCR, the mRNA expression of caspase-3 in SAH group was more than normal control, and in SAH+CLB group was the most. In pyridoxol group, the mRNA expression of caspase-3 was less than SAH+CLB group. The mRNA expression of Bcl-2 in SAH group was less than normal control, and in SAH+CLB group was the least. In pyridoxol group, the mRNA expression of Bcl-2 was more than SAH+CLB group.CONCLUSION:1. Pathological change of CVS after SAH was aggravated by blockage of cervical lymphatic drainage.2. Blood flow rate of microcirculation was degraded, and capillary vessel vasospasm was aggravated by blockage of cervical lymphatic drainage after SAH. 3. Pyridoxol can partly relieve CVS and abnormality of microcirculation by blockage of cervical lymphatic drainage after SAH.4. Brain edema after SAH was aggravated by blockage of cervical lymphatic drainage.5. Blockage of cervical lymphatic drainage can aggravated cerebral ischemic injury following SAH by down-regulation of Bcl-2 and up-regulation caspase-3.6. Pyridoxol can partly relieve the cerebral ischemic injury following SAH by blockage of cervical lymphatic drainage. |
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