淋巴性脑水肿大鼠的血压变化及其机制探讨
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摘要
脑水肿(brain edema,BE)是指脑组织含水量增加引起脑容积的扩张,为中枢神经系统对各种原因造成的脑损害产生的一种组织病理反应,是一个重要的病理生理过程。它是脑外伤、脑缺血或者缺氧的严重并发症,表现为脑容积扩大、颅内压增高,甚至发生脑病死亡,因而危害极其严重。脑水肿时液体的积聚可在细胞外间隙,也可在细胞膜内。前者称为血管源性脑水肿,多见于脑损伤、脑肿瘤等病变的初期,主要是由于毛细血管的通透性增加,导致水分在神经细胞和胶质细胞间隙潴留,促使脑体积增加所致;后者称为细胞中毒性脑水肿可能是由于某些毒素直接作用于脑细胞而产生代谢功能障碍,使钠离子和水分子潴留在神经细胞和胶质细胞内所致,但没有血管通透性改变,常见于脑缺血、脑缺氧的初期。我们研究发现尚存在另一种以大分子物质潴留为始动因素和主要特征的淋巴性脑水肿(lymphatic brain edema,LBE)。实验证实,脑内淋巴引流途径的受阻可引起神经细胞及胶质细胞外液增加,脑组织血浆蛋白等大分子物质及小分子物质潴留,血管周围间隙扩大并充满液体,即形成不同于以上两种水肿的LBE。当然这种分类方式有待商榷。
这种脑水肿与其他两种类型的脑水肿的关系如何?淋巴性脑水肿的机制何在?至今未见相关的报道。因此,我们采用手术阻断颈部淋巴引流的方法,建立LBE的模型,进而研究淋巴性脑水肿的发生、发展过程,探索其发生机制,为进一步研究淋巴性脑水肿相关的疾病治疗提供理论依据。迄今为止,虽尚未在脑内发现衬有内皮细胞的淋巴管结构,但已有确凿的证据证实了淋巴引流的存在。脑的淋巴引流不仅参与脑脊液的循环,而且担负着脑脊液及脑组织间隙液中蛋白质等大分子物质的重吸收,其在维持脑组织内环境稳态、颅内压稳定以及脑的正常生理功能等方面发挥着重要作用。
一方面,在临床上LBE见于头颈部急性、慢性炎症,喉癌、甲状腺癌、唇癌、牙龈癌、唾液腺肿瘤等行颈部淋巴结清扫手术,以及颈部外伤、气管切开、食道上段肿瘤的放疗等损伤颈部淋巴管及淋巴结,造成脑淋巴回流受阻。但由于其症状及体征缺乏特异性,长期以来相当一部分病人被误诊为“假性脑瘤”、“耳源性脑积水”、“原发性急性脑病”等,往往延误了治疗。因而研究LBE具有非常重要的临床意义。另一方面,淋巴性脑水肿大鼠在颈部淋巴结摘除后,颅内淋巴引流受阻,产生颅内压增高,形成单纯性的脑水肿模型。因为其排除机体的心脑血管基础病变及脑缺血缺氧的病理情况,研究BE这一病理生理过程的发生机制,对临床有效治疗各种脑部疾病共同存在的BE提供一条崭新的途径。
近十年来,泰山医学院脑微循环研究室在脑淋巴引流的生理和病理生理意义等方面做了积极的探索。主要工作在于脑淋巴引流途径及引流方式,淋巴性脑水肿后脑组织形态结构及功能的改变。研究表明,LBE引起了一系列生理指标,如皮层局域脑血流量、体感诱发电位、脑电图及颈淋巴压的改变。然而,LBE形成后血压的变化及其产生机制的系统研究未见报道。以往实验表明麻醉状态下LBE后大鼠血压升高、心血管功能降低。但是麻醉药物本身就抑制血压调节功能,从而对血压产生影响,掩盖了LBE的真实作用。大鼠在不使用麻醉剂的情况下,即清醒自由活动状态下,LBE对清醒自由活动大鼠的血压的影响如何?为此,我们首次研究了LBE对清醒自由活动大鼠血流动力学参数的影响。
研究表明,正常情况下血压不是固定不变的,而是自发变化的。血压的调节是一个涉及多系统多环节的复杂过程。目前较为肯定的血压调节因素主要包括神经系统、肾素-血管紧张素系统、肾上腺素能系统、缓激肽-前列腺素系统、内皮源性血管活性物质以及血管加压素等。由于血压的神经调节是最主要的调节方式,是通过动脉压力感受性反射(arterial baroreflex,ABR)实现的,然而LBE对清醒自由活动大鼠的ABR是否产生影响及这种影响的结构基础尚未见系统研究报道。本实验首次进行了LBE对孤束核的结构和功能的影响的系统探讨。
水通道蛋白-4(aquaporin-4,AQP4)在正常大鼠脑内具有广泛分布,主要表达于与血脑屏障相关的毛细血管内皮细胞及其相连的胶质细胞,其次是脑室室管膜上皮细胞、脑室脉络丛上皮细胞和下丘脑神经元。脑干神经核团和大脑皮质部分神经元也有分布。AQP4是脑组织中表达最高的水孔蛋白,在脑内主要参与脑内水代谢以及全身水代谢的调节。众多文献已经证实,AQP4参与了脑缺血、脑出血及脑创伤后脑水肿的形成和消散。然而,淋巴性脑水肿形成机制中AQP4的地位尚不明确。为此,本实验首次研究了LBE与AQP4基因及蛋白表达的关系,明确了AQP4在LBE中的作用。
研究报道,心房钠尿肽(atrial natriuretic peptide,ANP)是肾素-血管紧张素系统(renin-angiotensin system,RAS)的天然内源性拮抗剂,二者在血压的体液调节中发挥了重要的作用。但LBE对ANP及血管紧张素(angiotoninⅡ,AngⅡ)血浆水平有无影响,二者是否参与了LBE这一病理生理过程中血压改变的作用尚未见报道。本实验首次研究了LBE对AngⅡ和ANP血浆含量水平的影响,探讨了其在淋巴性脑水肿所致血压变化中的作用。
研究目的
1.探讨LBE对清醒自由活动大鼠血压及血压调节的影响。
2.探讨孤束背内侧亚核的组织病理变化在LBE后血压及血压调节的改变中的作用。
3.探讨AQP4在LBE中的作用。
4.探讨血浆AngⅡ及ANP在LBE后血压及血压调节的改变中的作用。
通过这四部分实验研究,以期为深入阐明淋巴性脑水肿对清醒自由活动大鼠血压的影响提供较新的神经、体液的以及分子生物学实验依据。
材料与方法
1.淋巴性脑水肿动物模型的建立
选用健康成年雄性Sprague-Dawley大鼠,参照Casley-Smith创立的并由本实验室改进的方法建立淋巴性脑水肿动物模型。具体方法如下:大鼠在盐酸氯胺酮50 mg/kg和地西泮5 mg/kg腹腔注射麻醉下,取仰卧位固定,颈部正中切口,分离两侧的颈浅淋巴结(每侧各3～5个),结扎相应的淋巴管,并摘除颈浅淋巴结。然后进一步分离出两侧的颈深淋巴结(每侧各1～2个),同法结扎其两端的淋巴管,并摘除颈深淋巴结,阻断颈部淋巴引流,造成淋巴性脑水肿。
2.动物分组
动物随机分为以下三组:(1)正常组(Normal组):仅进行股动、静脉插管进行血压监测,不接受颈部手术,不形成淋巴性脑水肿。(2)淋巴性脑水肿假手术组(Sham组):只进行颈部假手术,即分离颈部的淋巴管及淋巴结,但不结扎淋巴管,也不摘除淋巴结,不形成淋巴性脑水肿。(3)淋巴性脑水肿模型组(LBE组):进行颈部手术,分离颈部的淋巴管及淋巴结,结扎淋巴管,并且摘除淋巴结造成淋巴性脑水肿。
3.检测指标与方法
(1)应用清醒动物血压监测技术观察淋巴性脑水肿术后清醒自由活动大鼠血压、心率、血压波动性、心率变异性及压力反射敏感性随时间的变化规律。
(2)应用透射电镜技术观察LBE后孤束核背内侧亚核(dorsomedial nucleusof the solitary tract,dmNTS)病理组织学改变。应用抗神经元抗体免疫组化染色检测LBE后dmNTS内主要参与血压调节的神经递质谷氨酸(glutamic acid,GLU)及γ-氨基丁酸(gamma aminobutyric acid,GABA)表达的改变。应用抗神经元抗体免疫荧光染色dmNTS内谷氨酸脱羧酶67(glutamic aciddecarboxylase-67,GAD67)表达的改变。应用RT-PCR及Western blotting技术检测LBE对孤束核(nucleus of the solitary tract,NTS)内GAD67信使核糖核酸(mRNA)及蛋白表达的改变。
(3)应用干湿重法检测LBE对脑干脑水含量改变。应用RT-PCR技术检测LBE对NTS内水通道蛋白AQP4 mRNA表达的改变。应用抗神经元抗体免疫荧光染色技术检测各时间点dmNTS的AQP4蛋白表达的的变化。应用Westernblotting技术,检测LBE对NTS内AQP4蛋白表达的影响。并将AQP4的表达与脑水含量进行相关分析。
(4)应用特异性放射免疫均相竞争分析法(radioimmunoassay,RIA),检测LBE术后大鼠各时间点血浆血管活性物质血管紧张素Ⅱ及心房钠尿肽含量水平,并与血压作相关分析。
4.统计学处理
实验数据用均数±标准差((?)±s)表示。各组间比较采用单因素方差分析和多重比较检验。P＜0.05认为有显著性差异。
结果
1.LBE对清醒自由活动大鼠血压及血压调节功能的影响
假手术组各参数与正常组比较无统计学意义。与假手术组相比,LBE术后第1天SBP、DBP、MAP、HR开始下降(P＜0.05),第7天降至最低(P＜0.01),其数值分别为111.10±10.79 mmHg,70.46±9.98 mmHg,84.00±7.56 mmHg和324.06±18.06 bpm,至21天恢复正常。相反,SBPV、DBPV及HRV则在LBE术后第1天出现显著升高(P＜0.01),第7天升至最高点(P＜0.01),与假手术组相比分别增加56.65%,69.10%and 40.95%,后逐渐降低,至21天恢复正常。BRS变化趋势与血压、心率趋势相同,与假手术组相比,LBE后第1、7和15天分别降低23.81%,30%和23.53%(P＜0.01)。
2.LBE对孤束核背内侧亚核神经细胞病理组织学的影响
透射电镜超微结构观察发现,LBE后神经元细胞膜、内质网、线粒体膜、核膜不清晰、水肿,细胞质少,并出现裂隙、大液泡,核染色深,神经元异染色质显著增多、边集。胶质细胞膜水肿,周围出现空泡变性。髓鞘变性坏死,血管内皮细胞膜水肿,并出现周围裂隙。以LBE7天变化显著。免疫组织化学观察发现LBE后1天Glu免疫组化阳性染色细胞增多,至7天阳性神经元数目最多和染色强度最高,以后逐渐恢复。LBE后1天GABA免疫组化阳性染色细胞即见减少,至7天阳性神经元数目最少,免疫染色强度最低,以后逐渐改善。
3.LBE对孤束核GAD表达的影响
免疫荧光结果显示,假手术组和正常组大鼠dmNTS神经元GAD67蛋白表达无显著性差异。与假手术组比较,LBE组大鼠自术后1天GAD67免疫荧光阳性染色细胞即明显增多,以LBE7天最显著(P＜0.01),21天恢复正常。RT-PCR结果显示,LBE术后1天NTS组织就有较假手术组高的GAD67 mRNA表达(P＜0.05),7天达到表达的高峰(P＜0.01),Sham组和正常组GAD67表达水平相似(P＞0.05)。Western blotting结果显示GAD67蛋白的表达与mRNA表达呈现相同的趋势,在LBE术后7天升高94.7%。
4.LBE对脑干脑水含量及孤束核AQP4表达的影响
脑干水含量测定结果显示,正常组和假手术组大鼠脑含水量在各个时点无明显差异(P＞0.05)。与假手术组同一时间点相比,LBE组大鼠自术后1天脑组织含水量明显升高,7天达高峰,后逐渐恢复。
免疫荧光结果显示,正常组和假手术组均可见少量AQP4蛋白表达阳性细胞,两者无显著性差异(P＞0.05)。RT-PCR及Western blotting结果显示,孤束核部位AQP4的表达,正常组和假手术组相比差别无显著性。在淋巴性脑水肿组,在术后第1天表达水平开始上升(P＜0.05),7天达到峰值(P＜0.01),21d恢复到正常水平(P＞0.05)。脑干脑水含量和AQP4 mRNA及蛋白表达均呈现密切相关关系(mRNA:r=0.9452,P＜0.01;蛋白:r=0.8102,P＜0.01)。
5.LBE对血浆中AngⅡ及ANP含量水平的影响
与假手术组相比较,LBE大鼠血浆ANP含量显著升高(分别为P＜0.05和P＜0.01),峰值出现在7天。AngⅡ含量显著降低(分别为P＜0.05和P＜0.01),谷值出现在7天。正常组和假手术组大鼠血浆ANP和AngⅡ含量在各个时点无明显差异。相关分析显示,AngⅡ和ANP含量与LBE大鼠血压均呈显著正相关(r=0.9584,P＜0.01)和显著负相关(r=-0.9103,P＜0.01)
1.淋巴性脑水肿可导致清醒自由活动大鼠血压及血压调节功能的降低。
2.淋巴性脑水肿对孤束核背内侧亚核组织结构产生明显的损伤。
3.淋巴性脑水肿上调孤束核背内侧亚核神经的元谷氨酸的表达,下调γ-氨基丁酸及谷氨酸脱羧酶的表达,增加兴奋性氨基酸的神经毒性作用,从而恶化脑损害,最终抑制血压的神经调节功能,血压降低。
4.脑干脑水含量在淋巴性脑水肿大鼠明显增加,并通过上调水通道蛋白-4的表达实现的。
5.淋巴性脑水肿能诱导血管紧张素Ⅱ和心房钠尿肽血浆含量水平的变化,从而实现其血压降低的作用。
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.
Objective
1. 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.
Methods
1. Lymphatic brain edema model
Male 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. Groups
Rats 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 analysis
The 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.
Results
1. Changes in hemodynamic parameters and ABR in rats with LBE
The 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 dmNTS
Observation 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 NTS
Data 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 7~(th) 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 NTS
The 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 plasma
LBE resulted in a greater ANP content in plasma than did sham operation (P<0.05 at 1d and P<0.01 at the 7~(th) 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.
Conclusions
1. 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.
这种脑水肿与其他两种类型的脑水肿的关系如何?淋巴性脑水肿的机制何在?至今未见相关的报道。因此,我们采用手术阻断颈部淋巴引流的方法,建立LBE的模型,进而研究淋巴性脑水肿的发生、发展过程,探索其发生机制,为进一步研究淋巴性脑水肿相关的疾病治疗提供理论依据。迄今为止,虽尚未在脑内发现衬有内皮细胞的淋巴管结构,但已有确凿的证据证实了淋巴引流的存在。脑的淋巴引流不仅参与脑脊液的循环,而且担负着脑脊液及脑组织间隙液中蛋白质等大分子物质的重吸收,其在维持脑组织内环境稳态、颅内压稳定以及脑的正常生理功能等方面发挥着重要作用。
一方面,在临床上LBE见于头颈部急性、慢性炎症,喉癌、甲状腺癌、唇癌、牙龈癌、唾液腺肿瘤等行颈部淋巴结清扫手术,以及颈部外伤、气管切开、食道上段肿瘤的放疗等损伤颈部淋巴管及淋巴结,造成脑淋巴回流受阻。但由于其症状及体征缺乏特异性,长期以来相当一部分病人被误诊为“假性脑瘤”、“耳源性脑积水”、“原发性急性脑病”等,往往延误了治疗。因而研究LBE具有非常重要的临床意义。另一方面,淋巴性脑水肿大鼠在颈部淋巴结摘除后,颅内淋巴引流受阻,产生颅内压增高,形成单纯性的脑水肿模型。因为其排除机体的心脑血管基础病变及脑缺血缺氧的病理情况,研究BE这一病理生理过程的发生机制,对临床有效治疗各种脑部疾病共同存在的BE提供一条崭新的途径。
近十年来,泰山医学院脑微循环研究室在脑淋巴引流的生理和病理生理意义等方面做了积极的探索。主要工作在于脑淋巴引流途径及引流方式,淋巴性脑水肿后脑组织形态结构及功能的改变。研究表明,LBE引起了一系列生理指标,如皮层局域脑血流量、体感诱发电位、脑电图及颈淋巴压的改变。然而,LBE形成后血压的变化及其产生机制的系统研究未见报道。以往实验表明麻醉状态下LBE后大鼠血压升高、心血管功能降低。但是麻醉药物本身就抑制血压调节功能,从而对血压产生影响,掩盖了LBE的真实作用。大鼠在不使用麻醉剂的情况下,即清醒自由活动状态下,LBE对清醒自由活动大鼠的血压的影响如何?为此,我们首次研究了LBE对清醒自由活动大鼠血流动力学参数的影响。
研究表明,正常情况下血压不是固定不变的,而是自发变化的。血压的调节是一个涉及多系统多环节的复杂过程。目前较为肯定的血压调节因素主要包括神经系统、肾素-血管紧张素系统、肾上腺素能系统、缓激肽-前列腺素系统、内皮源性血管活性物质以及血管加压素等。由于血压的神经调节是最主要的调节方式,是通过动脉压力感受性反射(arterial baroreflex,ABR)实现的,然而LBE对清醒自由活动大鼠的ABR是否产生影响及这种影响的结构基础尚未见系统研究报道。本实验首次进行了LBE对孤束核的结构和功能的影响的系统探讨。
水通道蛋白-4(aquaporin-4,AQP4)在正常大鼠脑内具有广泛分布,主要表达于与血脑屏障相关的毛细血管内皮细胞及其相连的胶质细胞,其次是脑室室管膜上皮细胞、脑室脉络丛上皮细胞和下丘脑神经元。脑干神经核团和大脑皮质部分神经元也有分布。AQP4是脑组织中表达最高的水孔蛋白,在脑内主要参与脑内水代谢以及全身水代谢的调节。众多文献已经证实,AQP4参与了脑缺血、脑出血及脑创伤后脑水肿的形成和消散。然而,淋巴性脑水肿形成机制中AQP4的地位尚不明确。为此,本实验首次研究了LBE与AQP4基因及蛋白表达的关系,明确了AQP4在LBE中的作用。
研究报道,心房钠尿肽(atrial natriuretic peptide,ANP)是肾素-血管紧张素系统(renin-angiotensin system,RAS)的天然内源性拮抗剂,二者在血压的体液调节中发挥了重要的作用。但LBE对ANP及血管紧张素(angiotoninⅡ,AngⅡ)血浆水平有无影响,二者是否参与了LBE这一病理生理过程中血压改变的作用尚未见报道。本实验首次研究了LBE对AngⅡ和ANP血浆含量水平的影响,探讨了其在淋巴性脑水肿所致血压变化中的作用。
研究目的
1.探讨LBE对清醒自由活动大鼠血压及血压调节的影响。
2.探讨孤束背内侧亚核的组织病理变化在LBE后血压及血压调节的改变中的作用。
3.探讨AQP4在LBE中的作用。
4.探讨血浆AngⅡ及ANP在LBE后血压及血压调节的改变中的作用。
通过这四部分实验研究,以期为深入阐明淋巴性脑水肿对清醒自由活动大鼠血压的影响提供较新的神经、体液的以及分子生物学实验依据。
材料与方法
1.淋巴性脑水肿动物模型的建立
选用健康成年雄性Sprague-Dawley大鼠,参照Casley-Smith创立的并由本实验室改进的方法建立淋巴性脑水肿动物模型。具体方法如下:大鼠在盐酸氯胺酮50 mg/kg和地西泮5 mg/kg腹腔注射麻醉下,取仰卧位固定,颈部正中切口,分离两侧的颈浅淋巴结(每侧各3～5个),结扎相应的淋巴管,并摘除颈浅淋巴结。然后进一步分离出两侧的颈深淋巴结(每侧各1～2个),同法结扎其两端的淋巴管,并摘除颈深淋巴结,阻断颈部淋巴引流,造成淋巴性脑水肿。
2.动物分组
动物随机分为以下三组:(1)正常组(Normal组):仅进行股动、静脉插管进行血压监测,不接受颈部手术,不形成淋巴性脑水肿。(2)淋巴性脑水肿假手术组(Sham组):只进行颈部假手术,即分离颈部的淋巴管及淋巴结,但不结扎淋巴管,也不摘除淋巴结,不形成淋巴性脑水肿。(3)淋巴性脑水肿模型组(LBE组):进行颈部手术,分离颈部的淋巴管及淋巴结,结扎淋巴管,并且摘除淋巴结造成淋巴性脑水肿。
3.检测指标与方法
(1)应用清醒动物血压监测技术观察淋巴性脑水肿术后清醒自由活动大鼠血压、心率、血压波动性、心率变异性及压力反射敏感性随时间的变化规律。
(2)应用透射电镜技术观察LBE后孤束核背内侧亚核(dorsomedial nucleusof the solitary tract,dmNTS)病理组织学改变。应用抗神经元抗体免疫组化染色检测LBE后dmNTS内主要参与血压调节的神经递质谷氨酸(glutamic acid,GLU)及γ-氨基丁酸(gamma aminobutyric acid,GABA)表达的改变。应用抗神经元抗体免疫荧光染色dmNTS内谷氨酸脱羧酶67(glutamic aciddecarboxylase-67,GAD67)表达的改变。应用RT-PCR及Western blotting技术检测LBE对孤束核(nucleus of the solitary tract,NTS)内GAD67信使核糖核酸(mRNA)及蛋白表达的改变。
(3)应用干湿重法检测LBE对脑干脑水含量改变。应用RT-PCR技术检测LBE对NTS内水通道蛋白AQP4 mRNA表达的改变。应用抗神经元抗体免疫荧光染色技术检测各时间点dmNTS的AQP4蛋白表达的的变化。应用Westernblotting技术,检测LBE对NTS内AQP4蛋白表达的影响。并将AQP4的表达与脑水含量进行相关分析。
(4)应用特异性放射免疫均相竞争分析法(radioimmunoassay,RIA),检测LBE术后大鼠各时间点血浆血管活性物质血管紧张素Ⅱ及心房钠尿肽含量水平,并与血压作相关分析。
4.统计学处理
实验数据用均数±标准差((?)±s)表示。各组间比较采用单因素方差分析和多重比较检验。P＜0.05认为有显著性差异。
结果
1.LBE对清醒自由活动大鼠血压及血压调节功能的影响
假手术组各参数与正常组比较无统计学意义。与假手术组相比,LBE术后第1天SBP、DBP、MAP、HR开始下降(P＜0.05),第7天降至最低(P＜0.01),其数值分别为111.10±10.79 mmHg,70.46±9.98 mmHg,84.00±7.56 mmHg和324.06±18.06 bpm,至21天恢复正常。相反,SBPV、DBPV及HRV则在LBE术后第1天出现显著升高(P＜0.01),第7天升至最高点(P＜0.01),与假手术组相比分别增加56.65%,69.10%and 40.95%,后逐渐降低,至21天恢复正常。BRS变化趋势与血压、心率趋势相同,与假手术组相比,LBE后第1、7和15天分别降低23.81%,30%和23.53%(P＜0.01)。
2.LBE对孤束核背内侧亚核神经细胞病理组织学的影响
透射电镜超微结构观察发现,LBE后神经元细胞膜、内质网、线粒体膜、核膜不清晰、水肿,细胞质少,并出现裂隙、大液泡,核染色深,神经元异染色质显著增多、边集。胶质细胞膜水肿,周围出现空泡变性。髓鞘变性坏死,血管内皮细胞膜水肿,并出现周围裂隙。以LBE7天变化显著。免疫组织化学观察发现LBE后1天Glu免疫组化阳性染色细胞增多,至7天阳性神经元数目最多和染色强度最高,以后逐渐恢复。LBE后1天GABA免疫组化阳性染色细胞即见减少,至7天阳性神经元数目最少,免疫染色强度最低,以后逐渐改善。
3.LBE对孤束核GAD表达的影响
免疫荧光结果显示,假手术组和正常组大鼠dmNTS神经元GAD67蛋白表达无显著性差异。与假手术组比较,LBE组大鼠自术后1天GAD67免疫荧光阳性染色细胞即明显增多,以LBE7天最显著(P＜0.01),21天恢复正常。RT-PCR结果显示,LBE术后1天NTS组织就有较假手术组高的GAD67 mRNA表达(P＜0.05),7天达到表达的高峰(P＜0.01),Sham组和正常组GAD67表达水平相似(P＞0.05)。Western blotting结果显示GAD67蛋白的表达与mRNA表达呈现相同的趋势,在LBE术后7天升高94.7%。
4.LBE对脑干脑水含量及孤束核AQP4表达的影响
脑干水含量测定结果显示,正常组和假手术组大鼠脑含水量在各个时点无明显差异(P＞0.05)。与假手术组同一时间点相比,LBE组大鼠自术后1天脑组织含水量明显升高,7天达高峰,后逐渐恢复。
免疫荧光结果显示,正常组和假手术组均可见少量AQP4蛋白表达阳性细胞,两者无显著性差异(P＞0.05)。RT-PCR及Western blotting结果显示,孤束核部位AQP4的表达,正常组和假手术组相比差别无显著性。在淋巴性脑水肿组,在术后第1天表达水平开始上升(P＜0.05),7天达到峰值(P＜0.01),21d恢复到正常水平(P＞0.05)。脑干脑水含量和AQP4 mRNA及蛋白表达均呈现密切相关关系(mRNA:r=0.9452,P＜0.01;蛋白:r=0.8102,P＜0.01)。
5.LBE对血浆中AngⅡ及ANP含量水平的影响
与假手术组相比较,LBE大鼠血浆ANP含量显著升高(分别为P＜0.05和P＜0.01),峰值出现在7天。AngⅡ含量显著降低(分别为P＜0.05和P＜0.01),谷值出现在7天。正常组和假手术组大鼠血浆ANP和AngⅡ含量在各个时点无明显差异。相关分析显示,AngⅡ和ANP含量与LBE大鼠血压均呈显著正相关(r=0.9584,P＜0.01)和显著负相关(r=-0.9103,P＜0.01)
1.淋巴性脑水肿可导致清醒自由活动大鼠血压及血压调节功能的降低。
2.淋巴性脑水肿对孤束核背内侧亚核组织结构产生明显的损伤。
3.淋巴性脑水肿上调孤束核背内侧亚核神经的元谷氨酸的表达,下调γ-氨基丁酸及谷氨酸脱羧酶的表达,增加兴奋性氨基酸的神经毒性作用,从而恶化脑损害,最终抑制血压的神经调节功能,血压降低。
4.脑干脑水含量在淋巴性脑水肿大鼠明显增加,并通过上调水通道蛋白-4的表达实现的。
5.淋巴性脑水肿能诱导血管紧张素Ⅱ和心房钠尿肽血浆含量水平的变化,从而实现其血压降低的作用。
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.
Objective
1. 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.
Methods
1. Lymphatic brain edema model
Male 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. Groups
Rats 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 analysis
The 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.
Results
1. Changes in hemodynamic parameters and ABR in rats with LBE
The 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 dmNTS
Observation 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 NTS
Data 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 7~(th) 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 NTS
The 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 plasma
LBE resulted in a greater ANP content in plasma than did sham operation (P<0.05 at 1d and P<0.01 at the 7~(th) 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.
Conclusions
1. 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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