| Clinically, many pathologic conditions, such as cervical lymph-node removal of carcinoma of larynx, thyroid, lip, gingiva and salivary gland, tonsil resection, various kinds of inflammations in head and cervical part, as well as neck trauma, incision of tracheal and the radiotherapy on esophageal neoplasms of superior segment, may damage the cervical lymphatics and lymph nodes, and result in the blockade of cerebral lymphatic drainage. Lymphostatic hydrocephalus plays an important role in the human pathology. However, cerebral lymph drainage is neglected for a long time because the clinical manifestations in these patients are absent of speciality, which result in some patients to be misdiagnosed as other diseases, such as "pseudotumor cerebri", "otogenic hydrocephalus" or "benign intracranial hypertension", and the treatments to be usually delayed. As a result, it is meaningful to study cerebral lymphatic drainage.Many experiments have demonstrated that the lymphatic vessels in the neck provide the most important lymphatic pathway for CSF clearance. Lymph fluid in brain drains into extracranial lymphatic system by perineurolymphatics and prelymphatics pathways, which plays an important role in maintaining the normal physiological functions of brain and spinal cord. The blocking of this pathway can lead to the increase of extracellular fluid, and promote the retention of macromolecular material, such as plasma protein, which is known as "lymphatic brain edema" (LBE).In order to elucidate the mechanism, we establish lymphatic brain edema model by surgical blockade of the cervical lymphatic drainage to examine its development procedure and explore the changes of brain morphology and function. The LBE-induced alterations of many physiological indexes, such as cortex regional cerebral blood flow, somatosensory evoked potential, electroencephalogram and cervical lymphatic pressure, have been reported by this laboratory and others. However, the response of systemic arterial blood pressure to this pathophysiological process of lymphatic brain edema and the possible mechanism have not been systematically answered.Previous study showed that lymphatic brain edema lead to the elevation in blood pressure and depression in cardiovascular function in the anaesthetized rats. Frankly, the anaesthetics themselves do inhibit the blood pressure regulation and influence the level of blood pressure, which covers up the real action of LBE on systemic arterial blood pressure. It has not been reported that the effect of LBE on cardiovascular function in conscious animals. Consequently, the present work was designed to observe the variations of LBE on BP and HR in conscious unrestrained rats with LBE using a computerized hemodynamic monitoring system for the first time. In our system each rat was completely free from anesthetics, thus eliminating its blood pressure lowering and baroreflex function inhibitory effects.An attempt has also been made to interpret the mechanism involved in the above-mentioned variations from the three points of view, i.e., neuroregulation, humoral regulation and molecular biological basis.Generally speaking, BP is not consistent but undergoes spontaneous variations under physiological circumstances, further, its stability is chiefly regulated via arterial baroreflex (ABR) that acts as an effective buffer for BP fluctuations and prevents excessive BP swings. The baroreflex arc may be interrupted by complete lesion of the nucleus tractus solitarus. Hence, the NTS plays a key role in cardiovascular regulation, because baroreceptor and chemoreceptor afferent fibres terminate in the location, particularly the dorsomedial nucleus of the solitary tract (dmNTS) is preferentially barosensitive. The action of LBE on ABR and the structure in dmNTS haven't yet been investigated. This study was performed to answer these questions.Aquaporin-4 (AQP4) universally distributes in rats' brains, where it is expressed in capillary endothelial cells related to blood-brain barrier and astrocyte foot processes near blood vessels, in ependymal and pial surfaces in contact with cerebrospinal fluid, as well as in neurons in hypothalamic nuclei, brain stem nuclei and partial cerebral cortex. AQP4 is the most abundant water channel in brain and plays a critical role in cerebral and systemic water homeostasis because of its exceptionally high intrinsic water permeability. As shown by a number of studies, AQP4 mediates the formation and dispersion of brain edema inducted by cerebral ischemia, hemorrhage and trauma. Whereas, the status of AQP4 water channels in the LBE course has not been clarified. The experiments was conducted to elucidate the relationship between LBE and the express in AQP4 and identify the role of AQP4 in this physiopathological development.Researchers belive that atrial natriuretic peptide (ANP) is intrinsic endogenous antagonist of renin-angiotensin system (RAS), moreover, both ANP and RAS play importent roles in BP regulation. However, whether LBE regulates the release of them and they participate in the LBE-induced BP variation are still unknown. In the present experiments, we studied for the first time the effect of LBE on the circulating levels of angiotensin II (AngII) and ANP, together with their roles and mechnisms in the hemodynamic changes.Objective1. To investigate if lymphatic brain edema alters the hemodynamic parameters and cardiovascular function in conscious freely moving rats and the possible primary mechanism.2. To study the role of histological alterations in dmNTS induced by LBE in the changes of hemodynamic parameters and cardiovascular function.3. To research the role of AQP4 in the LBE process. 4. To explore the action of AngII and ANP in blood plasma in the changes of hemodynamic parameters and cardiovascular function.Through the four sets of experiments, we aimed to provide new insights for further study of the neural, humoral and molecular biological mechenisms of the variations of LBE on blood pressure and heart rate.Methods1. Lymphatic brain edema modelMale adult Sprague-dawley rats were used. Based on the method of Casley-Smith and Foldi, the LBE model was performed with a slight modification of our previous studies. Briefly, the rats were anesthetized with a mixture of ketamine (50 mg/kg) and diazepam (5 mg/kg) administered intraperitoneally. To prevent respiratory congestion, atropine sulfate (0.4 mg/kg) was administered 10 mins prior to anesthesia. The cervical lymph nodes, including bilateral submandibular superficial and deep nodes, were identified and isolated under a dissecting microscope. The cervical lymphatic nodes were removed after obstructing their input and output tubes.2. GroupsRats were randomly divided into the following three groups:(1) Normal group: Rats were prepared by intubating in the femoral artery and vein for blood monitoring and drug administration, exempting from cervical operations which can produce lymphatic brain edema.(2) Sham group: Rats were prepared by performing sham operations, exposing the bilateral cervical lymph nodes and lymphatics, which were not occluded.(3) Lymphatic brain edema group (LBE group): Rats were prepared by performing cervical operations, exposing the bilateral cervical lymph nodes and lymphatics, and the cervical lymphatic nodes were removed after obstructing their input and output tubes, which can produce lymphatic brain edema.3. Parameters and techniques for study(1) With the monitoring of the hemodynamic parameters in conscious freely moving rats, the values of BP, HR, BPV, HRV and BRS were examined respectively in three groups. (2) With the techniques of HE staining, uranyl acetate staining and anti-NeuN immunohistochemical study, the pathological histology in the dmNTS after LBE were examined. Immunofluorescence staining was used for assay of the expression of GAD67 in the neurons in the dmNTS after LBE. RT-PCR, real time RT-PCR and Western blotting were applied to detect the expression of GAD67 mRNA and protein in the NTS after LBE respectively.(3) Dry-weight method was administrated to measure the effects of LBE on the water contents in the rats' brain stem. Immunofluorescence staining was adopted for evaluation of the expression of AQP4 in gliocytes in the dmNTS tissue after LBE. RT-PCR, real time RT-PCR and Western blotting were performed to inspect the expressions of AQP4 mRNA and protein in the NTS after LBE respectively. At last, correlation analyses were made between all the above expressions and the water contents.(4) Specific radioimmunoassay was used for estimation of the plasma levels of AngII and ANP in rats with LBE. Subsequently, correlation analyses were respectively conducted between the acquired values and BP.4. Statistical analysisThe values are expressed as mean±S.D. The statistical significance of differences between the mean values of groups was first determined by using one-way ANOVA and then the multiple comparison tests were performed. Differences with a value of P <0.05 were considered significant.Results1. Changes in hemodynamic parameters and ABR in rats with LBEThe results showed that the values of all the parameters are not significantly different in normal and sham-operated rats, whereas the values varied greatly at different time period of observation in rats from LBE group. For instance, the SBP, DBP, MAP and HR appeared to decrease significantly at day 1 after LBE operation (P<0.05 vs. sham-operated group), and their valley values occurred at 7 day in LBE group (111.10±10.79 mmHg, 70.46±9.98 mmHg, 84.00±7.56 mmHg and 324.06±18.06 bpm, respectively, P<0.01 vs. sham-operated group), then gradually increased after 7 day in LBE rats, and finally returned to the baseline at the 21st day. On the contrary, the values of SBPV, DBPV and HRV immediately increased from 1 day after LBE operation (P<0.01 vs. sham-operated group), moreover, the peak values occurred at 7 day in LBE group, with the increase of 56.65%, 69.10% and 40.95%, respectively, compared with those in sham-operated group (P<0.01). BRS significantly decreased at different time points in the LBE group compared with those in the sham-operated group (P<0.01) with the decreases of 23.81%, 30% and 23.53% at 1, 7 and 15 day, respectively. Moreover, the valley value appeared at 7 day after LBE operation.2. Histopathological changes in dmNTSObservation on ultrastructural changes in dmNTS in rats with LBE showed that the whole membrane system in neurons including endoplasmic reticulum, mitochondrial and nuclear membrane were damaged, notably mitochondria cristae were swollen, enlarged, turned lucent and disarrayed, fragmented and finally disappeared. In the gliocytes, the membrane edema occurred with periphery vacuolar degeneration, nuclear shrinkage, chromatin condensed, and void space was evident. Edematous fluid accumulated around microvessels, leading to the dilated and congested microvascular endothelium with partial breaks. In contrast to those of the sham-operated rats, the morphological changes in dmNTS appeared from 2 to 16 day after LBE operation and were most prominent in the 8 day in LBE rats.Data of immunohistoehemical staining showed that compared with the sham group , the frequency of expression of Glu in the neurons of the dmNTS region in the LBE group was greater (P<0.05), whereas the frequency of expression of GABA was significantly decreased (P<0.05). Their extremum value appeared in the 8 day after LBE operation.3. Effect of LBE on the expression of GAD67 in the NTSData of immunofluorescence staining showed that the expression of GAD67 in the neurons of the dmNTS region in the sham and normal groups was not obviously different. Compared with sham group, the frequeney of expression of GAD67 in the neurons of the dmNTS region in the LBE group was greater (P<0.05 at 1 d and P<0.01 at the 7th d, vs. sham group). According the results of RT-PCR and Western blotting examination, the expression of GAD67 in the neurons of the dmNTS region in the LBE group appeared the same tendency as that of immunofluorescence staining. At the 7 day after LBE operation, the increases in mRNA and protein in LBE-induced NTS were 121.6% and 94.7%, respectively.4. Effect of LBE on the cerebral water content in brain stem and the expression of AQP4 in the NTSThe cerebral water contents in brain stem demonstrated no obvious changes in the normal and sham-operated groups at different time points (P>0.05). Compared with sham group, the cerebral water contents of the brain stem tissue significantly increased from 1 d after LBE operation (P < 0.05 vs. sham-operated group), arrived the maximum at 7 d, and lasted to 15 d (P < 0.01 vs. sham-operated).In normal and sham-operated groups, AQP4-fluorescent intensity of gliocytes and microvascular endothelial cells of the dmNTS were present and detectable at different time points. The obvious fluorescent-intensity increases of AQP4 were observed all through 15d after administrating LBE, which reached the peak value at 7 d (P<0.01) and recovered to the baseline at 21 d (P>0.05). There were the same variation trend in the expression of AQP4 mRNA and protein dased on the examination of RT-PCR and Western blotting techniques. Furthermore, correlation analysis revealed that the expression of AQP4 was intimately correlated to the cerebral water content.5. Effect of LBE on the levels of AngII and ANP in blood plasmaLBE resulted in a greater ANP content in plasma than did sham operation (P<0.05 at 1d and P<0.01 at the 7th d, vs. sham group), and the crest value took on the 7th day after LBE induction. Plasma AngII activity was increased only in the group subjected to cervical lymphatic blockade, which was the most remarkable at the 7th day after LBE. Correlation analysis showed that the level of AngII was negative correlated (r=-0.9584, P<0.01), whereas the level of ANP was positive correlated (r=0.9103, P<0.01) to blood pressure. Conclusions1. Lymphatic brain edema decreases the levels of blood pressure and heart rate, which is realized by depressed cardiovascular regulating function in the conscious freely moving rats.2. The apparent injury of the histological structures in rats' dmNTS induced by lymphatic brain edema is the neural source of the cardiovascular dysfunction.3. Lymphatic brain edema is capable of upregulating the expression of glutamic acid and downregulating the expressions of gamma aminobutyric acid and glutamate decarboxylase in neurons in the rats' dmNTS region, then increasing the excitatory amino acid neurotoxicity and worsening neuronal injure. The final result is to inhibit neural regulatory function to lower blood pressure and heart.4. The water contents in the rats' brain stem after lymphatic brain edema induction significantly raise, which result from the upregulated expression of AQP4.5. Lymphatic brain edema may induce the changes in the contents of AngII and ANP in circulating blood plasma, which fulfils their humoral regulation on blood pressure in the conscious freely moving rats.
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