Effects Of Lymphostatic Encephalopathy-induced Brain Injury On Autonomic Nervous Regulating Function And The Protection Of Hydroxysafflor Yellow A In Rats

The Cerebrospinal fluid (CSF) maintains electrolyte and acid-base balance in central nervous system (CNS) and furnishes the neurons and glial cells. It also transports and removes the cell metabolites, neurotransmitters, releasing factors, neuropeptide, hormone and cerebral plasma proteins. 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. Previous studies have proved that brain lymph is drained by per neural lymphatic and perivascular lymphatic, 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. As a result, it may cause Lymphatic Brain Edema (LBE) and Lymphatic Encephalopathy (LE). The excision of cervical lymph nodes and radiation therapy during cancer treatment is the primary cause of the Lymphatic Encephalopathy. Human LE shows symptoms such as reduction of locomotor activity, apathia, hypomnesia, headache, dizziness, nausea, vomiting, and convulsion.With the increase in aging population and the severity of pollution, the incidence of cancer has been increasing, so is the number of patients having cervical lymphatic diseases resulted from cancer treatment. Due to limited knowledge on LE, misdiagnoses are not uncommon. In addition, ischemia and hypoxia of cerebral structure due to LE would impact its regulatory function to tissue and organs. In recent years, scholars have found that LE rats present alteration of autonomic nervous function, however, the mechanism of which is still unknown. The lack of knowledge of LE also leads to failing in its treatment. Consequently, fully understanding of the occurrence, development and pathology of LE, and discovering effective therapeutic method for LE will possess great significance.Hydroxysafflor yellow A is yellow amorphous powder which is extracted from the safflower. It has a significant protective effect against the neurological impairment caused by hypoxia and ischemia, and it is widely used for treatment of cerebrovascular and cardiovascular diseases. The underlying mechanisms involve the reduction in the lipid peroxidation, the suppression of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, the up-regulation of the expression of endothelial nitric oxide synthase (eNOS) protein, and the decrease of cell apoptosis and the structural damages at the nervous tissues. However, we are not aware whether HSYA could protect against LE-induced brain injury.Heart rate variability (HRV) is the minimum fluctuation of instantaneous heart period and it is widely used to indirectly assess the regulatory roles of the autonomic nervous system (ANS) in the cardiovascular function. Extracting and analyzing HRV signal can evaluate the contraction, proportion and influence to cardiovascular system, which are created by cardiac sympathetic nerve and cardiac vagus nerve. The common analytical indicators of HRV contain two parts, time-domain and frequency-domain respectively. Time-domain means analyzing the variation among R-R interval via tendency of dispersion approach. Frequency-domain indicator reflects contraction and proportion created by cardiac sympathetic nerve and cardiac vagus nerve, in which low frequency (LF) indicates cardiac sympathetic nerve intensity, high frequency (HF) indicates cardiac vagus nerve intensity, LF/HF indicates relative value between cardiac sympathetic nerve and cardiac vagus nerve. Although scholars have done some research on the cardiovascular regulation injury caused by LE, evaluation of the variability of heart rate thoroughly through a normal analysis tool has not yet been reported. Coronary heart disease, myocardial infarction is a common and frequently occurring disease of the elderly. In addition, aging itself can cause turbulence of lymphatic drainage and incidence of tumor is high among the older people.It has reported that rostral ventrolateral medulls (RVLM) take part in heart rate adjustment through the medium of neurotransmitter or neuromodulator. RVLM is relevant to neural discharge dominated respiratory rhythm and high frequency elements of HRV. But nobody has yet observed the structure change in the RVLM caused by LE and the research which related to its morphology change and heart rate variability has not been reported.The purpose of this study was to investigate LE-induced brain changes in rats by using a combination of behavioral assessments, histological, ECG recordings, real-time PCR and western blot techniques, and to study the protective effects of HSYA.Objective1. Comprehensive understand and evaluate the effects of the regulatory function of autonomic nervous system caused by LE.2. To observe the effects of shape structure in the rostral ventrolateral medullas, and to understand the damage situation and the extent of damage in this region;3. To investigate the relations between structure damage in the rostral ventrolateral medullas and changes of the heart rate variability;4. To investigate the protective effects of HSYA treatment against LE-induced brain injury;5. To discuss the protective function mechanism of hydroxyl safflower yellow A on the autonomic nervous system. Materials and Methods1. Animals and setting of groupsAdult male Wistar rats, weighing250-300g, were obtained from Experimental Animal Center, Shandong University, China. The total180rats were used in this study and randomly divided into three groups. Animals in SHAM-operated group (SHAM; n=60) were subjected to lymphatic nodes exposure surgery without the removal of lymphatic nodes and cervical lymphatic occlusion or any pharmacological treatment. The60rats in HSYA group were given5mg/kg HSYA via intraperitoneal injection once per day after operation. The other60rats in lymphostatic encephalopathy group (LE) received the same surgical procedures as animals in HSYA group but administered an equivalent dose of saline instead of HSYA. Each group was further divided into six subgroups according to the days1,3,5,7,14and21after operation. There were10animals in each subgroup.2. Induction of CLB rat modelsThe CLB model was induced by a modified method of Casley-smith. Briefly, the rats were anesthetized with pentobarbital sodium (30mg/kg) intraperitoneally. An incision of the skin in the neck was made along the midline, The bilateral shallow lymphatic nodes(3-5) 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(1-2) 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.3. Behavior analysisOn postoperative days1,3,5,7,14, and21, the behavior score of each experimental animals was evaluated respectively. Scoring method according to the modified cerebral ischemia model Lemay Lemay and Sun Y grading standards. 4. ECG recordings and analysis of HRV signalsWith0.3%sodium pentobarbital intraperitoneal anesthesia(1ml/100g) on postoperative days1,3,5,7,14and21respectively. The electrocardiogram (ECG)(limb lead â…¡) 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 â…¡ ECG was selected to be HRV analyzed using HRV analysis module. Extracting representative parameters include:the time-domain indices MEAN of HRV (the mean of all the RR 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 studyFor hematoxylin and eosin (H&E) staining, the RVLM were separated from three rats of each group after ECG measurement. And the RVLM was used for electron microscopy.6. RT-PCR and Western BlotRT-PCR and western blotting were used to test the eNOS expression in RVLM of LE rats, and the effects of HSYA on the eNOS expression.7. Statistical analysisSAS V9.0Chinese was used for statistical analysis. The positive normal distribution of all HRV parameters was tested by using UNIVARIATE procedure. The differences between LE and SHAM group, as well as LE and HSYA group at each time point were compared using one-way non-parameter test based on Wilcoxon score. Results1. Effects on rats behavioralAnimals in SHAM group displayed no behavior missing. The score of neurological deficit in LE group were significantly higher than that of the SHAM group (p<0.05), rats in LE group exhibited less grooming, drowsiness and less motility than those in SHAM group, which occurred on day3after the CLB operation, and returned to the normal on postoperative day21. However, animals treated with HSYA had lower neurological deficit scores, as compared with those in LE group(p<0.05).2. Effects on heart rate variability indicator and regulation of autonomic nervous system function.Time-domain indicator:MEAN:compared with SHAM section, LE section protracted remarkably since the third day after the surgical operation. There is no significant difference between HSYA and SHAM section. However, compared with LE section, there is significant difference after surgical operation3,5,7,14and21day respectively. HSYA showed recovery in various levels.SDNN:compared with SHAM, LE section protracted remarkably from the third day to the fourteen day after operation, However, HSYA protracted little, so there is significant difference between HSYA and LE section.RMSSD:compared with SHAM, LE section protracted remarkably from day1to day14after operation, and HSYA treatment can block the RMSSD increase owing to LE remarkably.Frequency-domain indicator:LF:There is no significant difference among HSYA SHAM and LE during time points.HF:LE section had been protracting remarkably from day3to day14, and achieves SHAM level at day21. HSYA section can balance out the HF increase created by LE during postoperation days, definitely from day5to day14.LF/HF:compared with SHAM, LE section descended remarkably from day3to day14. There is significant difference between HSYA and LE section, HSYA can ameliorate the diminution as a result of LE.3. Effects on morphology and structure of brain tissueHE 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. In contrast, neurons in LE group were sparsely distributed and irregularly arranged. Some neurons and glial cells were swollen and degenerated. Some had vacuoles around the neurons or in the cytoplasm. And the tunica adventitia of blood vessels was swollen and had interspace with cerebral tissue. These changes occurred on day3after CLB operation, and became obviously at postoperative day5and7, and then recovered gradually. HSYA treatment reduced LE-induced histopathological abnormalities.Ultrastructure observe:No apoptotic neurons were observed in SHAM group. In contrast, neurons in LE group showed that highly condensed nuclei, deeply stained chromatin, blurred nuclear membrane. In addition, the myelin sheath in LE group became loose or disintegrated. HSYA administration reduced the number of apoptotic neurons and reduced injury.4. Effects on eNOS expressionThe mRNA expression of eNOS was significantly decreased in LE group, as compared to that in SHAM group at postoperative day5and7(P<0.05), respectively. Treatment with HSYA significantly increased the mRNA expression of eNOS (P<0.05). This result was further confirmed by western blotting at protein levels. The eNOS protein expression in LE group was significantly down-regulated whereas up-regulated at each time point in HSYA-treated group. Conclusion1. LE can damage regulatory function of autonomic nervous system, reduce heart rate, increase heart rate variability and disturb the balance of Sympathovaga, causing vagus nerve dominance.2. LE can cause neuron, capillary and myelin sheath damaged in the RVLM.3. The structural damages of the RVLM appeared to be closely related to the changes of HRV at each time point. Thus, in LE rats, the dysfunction in the regulation of ANS might be due to the impaired structure in the RVLM.4. Treatment with HSYA can remarkably improve the regulatory function of ANS and the impaired structure in the RVLM and it can increase the LE-induced decreasing in eNOS mRNA and protein expression. Our study demonstrated that treatment with HSYA can effectively reduce the brain injury caused by LE and the mechanism might be partly attributed to the nitric oxide pathway.