| Antibodies, memory cells and antigen presenting cells cycle in the lymphoid organs tissue. The central nervous system is one of the systems that can minority itself which has the function of immune. The central nervous system does not exist lymphatic duct, that brain lymph fluid flow along the artery and blood capillary wall clearance. Cerebrospinal fluid is the environment of the brain, thus ensuring PH and osmotic pressure of cerebrospinal fluid normal is of great significance to ensure the steady of intracerebral internal environment, and maintain the normal structure and function of the brain.Previous studies have proved that brain lymph is drained by artery clearance, after which be drained extra-cranially via cervical lymph. Blocking any part of this pathway can result in turbulence of cerebrospinal fluid circulation and ventricular drainage, causing disturbance of internal environment of the nervous system and abnormal nerve function, leading to Lymphatic Encephalopathy (LE). The primary cause of the LE is the excision of cervical lymph nodes and radiation therapy during cancer treatment. The cardinal symptoms of human LE are reduction of locomotor activity, apathia, hypomnesia, headache, dizziness, nausea, vomiting, and convulsion.The incidence of cancer has been increasing, so does the number of patients with cervical lymphatic diseases resulted from cancer treatment. In addition, ischemia and hypoxia of cerebral structure due to LE would impact its regulatory function to tissue and organs. Recent studies showed that LE rats present alteration of autonomic nervous function, caused the blood pressure and heart rate adjustment ability dropped, body temperature significantly reduced. However, the mechanism of which is still unknown. Nitric oxide(NO) related to several central nervous system diseases, NO not only protect the normal metabolism of brain neurons, but also have cytotoxic effect on the brain neurons. Nitric oxide synthase(NOS) is the key enzyme for the synthesis of NO, inducible nitric oxide synthase(iNOS) can be synthesized in neurons, which is one of the important reason for the neuron injury. Hydroxysafflor yellow A are widely used in the treatment of heart and head blood-vessel diseases. But its application in the clinical treatment of lymphatic stranded encephalopathy records are rarely reported. To observe the microstructure and the inducible nitric oxide synthase expression of the prefrontal cortex neurons. To explore the protection of Hydroxysafflor yellow A on the prefrontal cortex nerve cells of LE rats.Objective1.To observe the prefrontal cortex neurons microstructure.2.To evaluate the three type nitric oxide synthase expression of experimental rats.3.To explore the Hydroxysafflor yellow A effect on the prefrontal cortex nerve cells of LE rats.Methods1.Animals and setting groupsTaking65Wistar rats, SPF level, weighing170-230g, the rats were separated into SHAM group, HSYA group and LE group randomly. Each group were respectively separated into1day,7days,14days three groups according to the postoperative time, total of nine groups, seven rats each group, accidental death in the process of experimental rats were supplemented by the rest of the rats.2.Establishment of LE models and treatment of the groupsThe rats of LE group and HSYA group were established by ligaturing lymphatic vessels and abating all superficial and internal cervical lymph nodes. The HSYA group rats were given Hydroxysafflor yellow A by intraperitoneal injection after the operation every day. The SHAM group were established by liberating lymphatic vessels and cervical lymph nodes. While other operating the same as the LE and HSYA group, and give the rats of SHAM and HSYA groups saline by intraperitoneal injection after the operation every day.3.Making paraffin section and dyeingPerfusing rats’ hearts using4%paraformaldehyde each group after1,7,14days of the operation, then make the paraffin sections using prefrontal cortex brain in order to view the expression of inducible nitric oxide synthase via immunohistochemical staining. In addition, take the prefrontal cortex brain without perusing heart for electron microscopy scanning.4.Histologic observation under transmission electron and optical microscopeObserve the PFC neurons and the change of the myelin sheath under transmission electron microscope. Observe the neurons changes under the optical microscope. View the expression of inducible nitric oxide synthase in immunohistochemical staining under optical microscope.5.Statistical analysisCollecting and analyzing data that obtained by IPP using SAS V9.3Chinese. Choose p<0.05for that difference was statistically significant.Resultsl.The ultrastructural of PFC neurons under optical microscopeThere are no evident difference between the groups of SHAM group. Showing clear neuron structure integrity, the cytoplasm is pink and well-distributed, cell nucleus is blue and its boundary is clear and oval, chromatin in nucleus color distributed evenly. There are no evident difference between the1day and7day group of HSYA and SHAM group, and the microscopic performance is similar to the SHAM group. However the the7day group of HSYA group can see boundaries fuzzy of cells and cell nuclei. In the LE group, permutation of nerve cells are disordered, cells shape are irregular, cell membrane boundary is not clear, the nuclei deepen and nuclear envelope breakdown. The most obvious microscopic performance is7day group of LE. While the figure of neurons damaged decreased in lday and14day groups of LE. 2.The neuron changes of PFC under electron microscopeThere are no evident difference among the day groups of SHAM group, which showing that, neurons structure is complete, orderly formed without apoptosis and necrosis phenomenon. There are no evident difference between1day and7day group of HSYA group, and the microscopic performance is similar to the SHAM group. The7day group of HSYA group neurons appear a certain degree of damage, but less than the LE group. All of the day groups of LE group show neurons injury,7day group is the worst. Appearing that nuclear chromatin, nucleolus boundaries fuzzy, nuclear chromosome crumb and cell dissociation.3.The myelin sheath changes of PFC under electron microscopeMyelin sheath is the mix of lipid and protein that wrapped around the neurons. Myelin sheath can protect neurons. Myelin sheath form structure is normal in the SHAM group. There are no evident difference between the1day and7day group of HSYA and SHAM group, and the microscopic performance is similar to the SHAM group. The7day groups of HSYA group myelin shows loosing curling and delamination. But no myelin fracture phenomenon. All of the day groups of LE group show myelin injury, and7day group is the worst, appearing seriously myelin loosing curling, delamination and myelin fracture.4.The nitric oxide synthase expressionThere are no evident difference between day group of SHAM group showing less of tan particle and the color is shallow. The average optical density value difference was statistically significant. There are evident difference between1day group of SHAM group and LE group(P<0.05), HSYA group and LE group(P<0.05). The1day group of LE average optical density value is the highest, and all the difference is evident(P<0.05).Conclusion1.Lymphatic fluid retention can lead to the prefrontal cortex nerve cells’form destroyed, the nuclear staining deepen and its envelop broken.2.Lymphatic fluid retention induced particular nerve cells synthesise amounts of inducible nitric oxide synthase, this may be associated with the damage of the prefrontal cortex neurons.3.Hydroxysafflor yellow A can restrain the expression of inducible nitric oxide and mitigate the damage. |
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