Nfulences Of Lymphostatic Encephalopathy ON The Ultrastructure And The Heart Rate Variability In Rats

The lymphatic drainage in the brain plays an important role in keeping the circulation of CSF and the balance of the internal environment of the brain[1]. Previous studies have proved that brain lymph is drained by perneural lymphatics and perivascular lymphatics, 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)[2]. 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 is 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. In recent years, the studies have found 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. Therefore, fully understanding of the occurrence, development and pathology of LE will have great significance.Heart rate variability (HRV) is a reflection of the autonomic nervous system (ANS) on the cardiovascular adjustment and is widely used to indirectly assess the regulatory roles of the autonomic nervous system (ANS) in the cardiovascular function. The rostral ventrolateral medulls (RVLM) is the important integration central of the sympathetic and cardiovascular function. RVLM accept all kinds of information of cardiovascular activity from other parts of the central and peripheral, and through descending pathways directly project to the sympathetic preganglionic neurons of the lateral thoracic segments of the spinal cord grey matter column, which regulates the sympathetic cardiovascular activities and has important influence to HRV.[4]. Temperature is important indicator to maintain the normal metabolism, the changes of temperature has the important influence on cardiovascular function.The purpose of this study was to investigate LE-induced changes of HVR, the changes of ultrastructure, the changes of temperature and eNOS protein expression in the RVLM of rats so as to understand the influence to the cardiovascular adjustment function, the temperature and the possible influence mechanism caused by LE and to discuss the relationship between the injury of RVLM and the changes of HRV.Objective1. To observe the influences of structure in the rostral ventrolateral medullas and to observe the damage situation in this region.2. To evaluate the influences of the regulatory function of autonomic nervous system caused by LE.3. To understand the influences of the temperature adjustment function caused by LE.4. To explore the correlation between HRV change and the temperature change caused by LE.5. To investigate the relations between injuries of RVLM and changes of the HRV.Materials and Methods1. Animals and setting of groupsAdult male Wistar rats, weighing250-300g, were obtained from Experimental Animal Center, Shandong University. The total80rats were used in this study and randomly divided into two groups, SHAM group and LE group. Each group was further divided into four subgroups according to the days1,7,14and21after operation. There were10animals in each subgroup.2. Induction of LE modelsReferring to literature, the LE model was induced by a modified method of blocking the neck lymph drainage. Procedures are as below:the rats were anesthetized with pentobarbital sodium (35mg/kg) intraperitoneally. An incision of the skin in the neck was made along the midline. The bilateral shallow lymphatic nodes were exposed and removed after ligating their afferent and efferent tubes. The trachea was exposed after splitting of subcutaneous tissue and musculature,until the trachea. The deep cervical lymphatic nodes could be found at the lateral to thyroid cartilage and posterolateral to the common carotid artery and the nervous vagus. The afferent and efferent lymphatic tubes were ligated, and the lymphatic nodes were then removed. Layered suture incisions. In SHAM group,the afferent and efferent lymphatic tubes were not ligated, and the lymphatic nodes were not removed. The rest of the surgical procedure was same as the experimental group.3. Behavior analysisOn the postoperative days1,7,14, and21, the behavior scores of each group experimental rats were evaluated respectively. The scoring method according to the modified cerebral ischemia model Lemay Lemay and Sun Y grading standards.4. ECG, temperature recordings and analysis of HRV signalsWith pentobarbital sodium (35mg/kg) intraperitoneally on postoperative days1,7,14and21respectively. The electrocardiogram (ECG)(limb lead II) was recorded with ECG sensor, the body temperature was monitored with temperature sensor. The signals were digitized at a sampling rate of2kHz and continuously recorded for30min using a AD Instrument PowerLab system. Then a duration of5-min stable signal was selected to be short-term HRV analyzed. The standard limb lead II ECG was selected to be HRV analyzed using HRV analysis module. Extracting representative parameters include:the time-domain indices:MEAN (the mean of all the R-R interval throughout)、 SDNN (the standard deviation of IBI of all data、 RMSSD (the square root of the mean of the sum of the squares of differences between successive IBI) and the frequency-domain indices:LF (the low frequency power,0.04<LF<0.15Hz); HF (the high frequency power,0.15<HF<0.4Hz) and LF/HF (ratio).5. Histological studyThe RVLM was separated from three rats of each group after ECG measurement, which for hematoxylin and eosin (H&E) staining, and the RVLM was used for electron microscopy observation.6. Western BlotWestern blotting were used to test the eNOS expression in RVLM of LE rats.7. Statistical analysisSAS V9.0Chinese was used for statistical analysis. The positive normal distribution of all HRV parameters were tested by using UNIVARIATE procedure. The differences between LE and SHAM group, as well as LE group at each time point were compared using one-way non-parameter test based on Wilcoxon score.ResultsI. Effects on rats behaviorThe rats in SHAM group displayed no behavior missing. Compared with SHAM group, scores of neurological deficit in LE group were significantly higher (p<0.05). The rats in LE group exhibited less grooming, drowsiness and less motility, which occurred on day3after LE operation, and returned to normal on the postoperative day21.2. Effects on heart rate variability indicator.Time-domain indicators:①Mean of inter-beats intervals (MEAN):LE group prolonged remarkably since the7day after LE operation(P<0.05). There were significant differences on the postoperative day7,14and21respectively, compared with SHAM group. The increase of MEAN explain the slow heart. ②Standard deviation of IBI of all data (SDNN):LE group increased significantly from the7day to the14day after LE operation (P<0.05), compared with SHAM. There was significant difference between SHAM group and LE group. That SDNN increase heart rate variability increases, cardiovascular regulating function decline.③Square root of the mean of the sum of the squares of differences between successive IBI (RMSSD):LE group prolonged remarkably on the postoperative day1,7,14(P<0.05), compared with SHAM group. The increase of RMSSD explain that the vagus nerve adjust function enhanced caused by LE.Frequency-domain indicators:①Low frequency power (LF):There was no significant difference between SHAM group and LE group during time points (P<0.05). Explained the influence of LE to sympathetic was not obvious.②High frequency power (HF):LE group prolonged remarkably from day7to day14, and returned to normal level at day21(P<0.05). HF increase reflected the vagus nerve adjust functions strengthened, and the change of RMSSD consistent.③LF/HF ratio (LF/HF):LE group reduced remarkably from day7to day14compared with SHAM group (P<0.05). There was significant difference between SHAM group and LE group. LF/HF is the important symbol of the vagus nerve balance. This ratio lower reflected the balance was destroyed, the regulating function of the vagus nerve predominant in the balance.3. Effects on morphology and structure of brainHE staining and light microscopy:In SHAM group, neurons and glial cells were distributed evenly with a clear boundary, the nucleus of neuron were large, round and lightly stained, nucleolus were clear and deeply stained. The blood vessels were normal. Compared with SHAM group, neurons in LE group were sparsely distributed and irregularly arranged. Some neurons were swollen and degenerated. There were vacuoles around the neurons or in the cytoplasm. And the vascular adventitia was swollen and had interspace with cerebral tissue. The changes occurred on day7after LE operation, and became obviously at postoperative day7and14, then recovered gradually.Ultrastructure observation:No apoptotic neurons were observed in SHAM group. The myelin structure was compact and arranged orderly. Vascular endothelial cells were normal. Compared with SHAM group, neurons in LE group showed that highly condensed nuclei, deeply stained chromatin, blurred nuclear membrane.The myelin sheath in LE group became loose or disintegrated. Capillary basement membrane hypertrophy. The half-moon-shaped or irregular shape expansion was observed in the outer membrane, which consisted a large number of edema fluid. The changes were most obvious on the postoperative day7.4. Effects on the temperatureResearches show that LE group, along with the development of LE, temperature had a trend of gradual drop. The temperature of LE group had negative relation to the disease process.(r=-0.42589,p=0.0005). The temperature in LE group dropped, as compared to that in SHAM group on postoperative day1and21, but not significant; There were significant differences between SHAM group and LE group on postoperative day7and day14(p<0.05); Further analysis showed that many parameters of temperature and heart rate variation had relationship in LE group. And the relationship did not exist in the SHAM group.5. Effects on eNOS expressionThe eNOS protein expression in LE group was significantly down-regulated, as compared to that in SHAM group at postoperative day7and14(P<0.05), respectively. This result was further confirmed by western blotting at protein levels.Conclusion1. LE damaged the adjust ability of autonomic nervous system. Performances were heart rate reduce, HRV increase. The balance of sympathetic and vagal system was broken, the vagus nerve dominated the balance.2. LE can damage the neurons, capillary and myelin sheath of the RVLM.3. LE damaged the body’s temperature adjustment ability, caused hypothermia.4. There is a correlation between the extent of the temperature drops with the changes of HRV.5. The changes of HRV appeared to be closely related to the structural damages of the RVLM at each time point. Thus the structure damage of RVLM caused by LE was closely related to the adjust ability of ANS.