Onset of aquaporin-4 expression in the developing mouse brain

The main water channel in the brain, aquaporin-4 (AQP4) is involved in maintaining homeostasis and water exchange in the brain. In adult mammalian brains, it is expressed in astrocytes, mainly, and in high densities in the membranes of perivascular and subpial endfeet. Here, we addressed the question how this polarized expression is established during development. We used immunocytochemistry against AQP4, zonula occludens protein-1, glial fibrillary acidic protein, and β-dystroglycan to follow astrocyte development in E15 to P3 NMRI mouse brains, and expression of AQP4. In addition we used freeze-fracture electron microscopy to detect AQP4 in the form of orthogonal arrays of particles (OAPs) on the ultrastructural level.We analyzed ventral, lateral, and dorsal regions in forebrain sections and found AQP4 immunoreactivity to emerge at E16 ventrally before lateral (E17) and dorsal (E18) areas. AQP4 staining was spread over cell processes including radial glial cells in developing cortical areas and became restricted to astroglial endfeet at P1–P3. This was confirmed by double labeling with GFAP. In freeze-fracture replicas OAPs were found with a slight time delay but with a similar ventral to dorsal gradient. Thus, AQP4 is expressed in the embryonic mouse brain starting at E16, earlier than previously reported. However a polarized expression necessary for homeostatic function and water balance emerges at later stages around and after birth.     

Fetal development of membrane water channel proteins aquaporin-1 and aquaporin-4 in the human brain

Aquaporin-1 and aquaporin-4, water channel membrane proteins reported in both experimental animals and in adult humans, have been detected in different, non-overlapping areas of the central nervous system. This immunohistochemical study describes the developmental expression pattern of the water channel membrane proteins, aquaporin-1 and aquaporin-4, in various structures of human fetal brain over the gestational period of 14-40 weeks. Aquaporin-1 immunostaining was exclusively found in the epithelial cells of the choroid plexus from the 14th gestational week, and the staining pattern altered slightly over time. At week 14, immunostaining appeared only in the apical cell membranes. By the 18th gestational week, the entire plasma membrane of these apical cells was immunopositive, as well as was the cytosol. These changes in immunoreactivity indicate an increasing production of aquaporin-1 in the epithelial cells during the period between the 14th and 24th weeks of gestation. Aquaporin-4 immunostaining was first detected in the archicortex, from gestational week 14 and was detected in the neocortex, 6-7 weeks later. Immunostained structures were always astrocytes, particularly the astrocytic endfeet in the ventricular wall, at the developing ependymal lining, at the pial surface, and around the capillaries. Neuronal labeling was not observed. These results in human fetal brain lend morphological support to the previous findings that aquaporin-1 and aquaporin-4 play different roles in the regulation of the water homeostasis of the brain.     

正常大鼠伽玛刀照射后脑内水通道蛋白-4的变化

目的 探索在放射性腩水肿中,水通道蛋白-4业型(Aquaporin-4,AQP4)的表达与脑水含量变化的关系.方法 使用Leksell C型伽玛刀分别以80Gy和160Gy的剂量对大鼠右侧基底节区进行照射,之后在1d、3d、7d及14d四个时间点用免疫组织化学法观察AQP4表达的变化,并使用干湿重法测定大鼠脑组织的水含量.结果 在80Gy组,脑水含量在3d达到高峰,并在14d时恢复到对照组水平,AQP4的表达分别在3d及14d两次明显升高;而在160Gy组,脑水含最和AQP4的表达在整个14d的观察期内均呈持续性增高.结论 AQP4在放射性脑水肿的形成及消散过程中均发挥了介导作用,其对水分子的跨膜转运可能是双向的.     

水通道蛋白4在大鼠蛛网膜下腔出血后脑水肿的表达研究

目的探讨大鼠蛛网膜下腔出血后脑水肿组织中水通道蛋白4（aquaproin4，AQP4）的表达变化。方法采用自体血注入枕大池法建立蛛网膜下腔出血动物模型，原位杂交和免疫组化法的方法分别检测蛛网膜下腔出血后AQP4mRNA和蛋白质表达变化，并用干湿重法测定脑含水量变化。结果蛛网膜下腔出血后8h，脑含水量和AQP4mRNA及蛋白表达开始上升，2d达到高峰，1周后降低到正常水平；AQP4 mRNA及蛋白的表达和脑含水量呈显著正相关（r=0．9236～0．9509，P〈0．01）。结论AQP4与蛛网膜下腔出血性脑水肿的形成密切相关。     

水通道蛋白-4在脑出血大鼠脑组织的分布

目的 观察脑出血大鼠水通道蛋白-4(AQP4)在脑组织的分布变化及其在出血性脑水肿中的作用。方法 采用定量胶原酶注人大鼠尾状核建立脑出血模型，应用免疫组化法观察脑出血后不同时间大鼠脑组织AQP4的表达。结果 AQP4阳性细胞主要分布在脑出血血肿周围区和大脑皮质的星形胶质细胞、脑室周围、视上核和室旁核。脑出血后6h起，AQP4表达增强，出血后72h达高峰，出血1周后仍高于正常水平。结论 脑出血后，AQP4在与水代谢密切相关的部位表达明显增强，提示AQP4在出血性脑水肿的形成过程中起重要作用。     

MPTP-induced neuroinflammation increases the expression of pro-inflammatory cytokines and their receptors in mouse brain

Parkinson's disease (PD) is a common neurodegenerative disease characterised by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Despite intensive research, the cause of neuronal loss in PD is poorly understood. Inflammatory mechanisms have been implicated in the pathophysiology of PD. In this study, conducted on an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we investigated the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and their receptors (IL-1RI, TNF-αRI, IL-6Rα) at the SN and caudate-putamen (CP) levels. In MPTP-treated animals we observed a significant increase in IL-1β, TNF-α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF-αRI and IL-6Rα were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD.     

水通道蛋白-4与脑膜瘤瘤周水肿的关系

目的 研究水通道蛋白-4(AQP-4)在脑膜瘤中的表达与瘤周水肿(PTBE)的关系.方法 选择脑膜瘤患者70例,计算水肿指数,免疫组化检测脑膜瘤中AQP-4、血管内皮生长因子(VEGF)表达水平,分析AQP-4、VEGF与脑膜瘤PTBE之间的关系.结果 本组脑膜瘤PTBE发生率62.9%(44/70).AQP-4和VEGF的表达阳性率在无水肿组分别为38.5%(10/26)和65.4%(17/26).而在水肿组则分别为90.9%(40/44)和93.2%(41/44);AQP-4 和VEGF 在脑膜瘤中的表达水平,两组间相差显著(P<0.01).AQP-4和VEGF表达水平均与脑膜瘤PTBE的程度呈正相关(P<0.05).结论 AQP4、VEGF在脑膜瘤中的高表达可能参与了其PTBE的形成.     

AQP-9在大鼠感染性脑水肿脑组织中的表达及意义

目的 探讨水通道蛋白-9(AQP-9)在内毒素脂多糖(LPS)致大鼠感染性脑水肿脑组织中的表达及意义. 方法 1月龄普通级SD大鼠128只采用随机数字表法分为生理盐水(NS)组(64只)和LPS组(64只),采用颈内动脉注射LPS制作大鼠感染性脑水肿模型,模型成功后每组均选取6h、12h、24h和48 h4个时间点,在不同时间点采用HE染色观察脑组织形态学改变;干湿重法测定脑组织含水量(BWC);甲酰胺法测定脑组织伊文思蓝(EB)含量;免疫组织化学法检测脑组织AQP-9蛋白的表达量:采用逆转录多聚酶链反应(RT-PCR)方法 检测AQP-9 mRNA的表达水平并对结果 进行相关性分析. 结果 HE染色结果 显示LPS组血管周围间隙增宽、炎性细胞浸润、胶质细胞体积增大肿胀、神经元空泡变性、细胞核固缩等.与NS组相比,LPS组6 h、12 h、24 h和48 hBWC、EB含量、AQP.9蛋白及AQP.9mRNA表达水平均增高.差异具有统计学意义(P<0.05).同时LPS组BWC和EB含量、AQP-9蛋白、AQP.9 mRNA表达量、AQP-9 mRNA表达量与EB含量、AQP-9蛋白与mRNA表达量均呈正相关. 结论 AQP-9可能参与感染性脑水肿的发生和发展.     

Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

INTRODUCTIONAquaporin (AQP), found recently, is a group of water permeability-re-lated cell membrane transport protein. Among them, AQP-4 is mostnoticeable. To suppress AQP-4 expression can relieve the formationof brain edema, and then reduce the death ra…     

Immunological identification of the mammalian H3 histamine receptor in the mouse brain

Affinity-purified antibodies raised against the peptide sequence H3 (349-358) receptor specifically recognized two protein species with Mr 62,000 and 93,000 in adult mouse forebrain membranes. Both immunoreactive species were suppressed greatly by preincubation of the antibody with the respective peptide. Immunohistochemical analysis using affinity-purified anti-H3 (349-358) antibodies yielded a high degree of coincidence with ligand-autoradiographical information, with high levels detected in the CA3 and dentate gyrus of the hippocampus, laminae V of the cerebral cortex, the olfactory tubercle, Purkinje cell layer of the cerebellum, substantia nigra, globus pallidus, thalamus and striatum. This study suggests further biochemical evidence for multiple H3 receptor subtypes and the widespread distribution of the H3 receptor in the mammalian brain.     

Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia.

Aquaporin-9 (AQP9) is a new member of the aquaporin family of water-selective channels mainly expressed in liver and testis, presenting the characteristic of also being permeable to various solutes, particularly lactate. Recent data have shown the presence of AQP9 on tanycytes in the rat brain. In the current study, the authors show the expression of AQP9 in astrocytes in the mouse brain and changes in its expression after cerebral ischemia. Indeed, in control mouse, the AQP9 immunolabeling is present on astrocytic processes bordering the subarachnoid space and ventricles. The labeling also is observed on astrocytes in the white matter, hippocampus, hypothalamus, and lateral septum. After focal transient ischemia, an increase of the immunolabeling is detected on astrocytes in periinfarct areas. This AQP9 distribution study in mouse brain suggests a role of AQP9 in water homeostasis in the central nervous system. Furthermore, the overexpression of AQP9 on astrocytes surrounding an ischemic lesion suggests that AQP9 may also play a role in the regulation of postischemia edema and, in view of its permeability to monocarboxylates, in the clearance of lactate from the ischemic focus.     

Redistribution of aquaporin-4 in human glioblastoma correlates with loss of agrin immunoreactivity from brain capillary basal laminae

Vasogenic edema is one of the most serious clinical problems in brain tumors and tightly connected to water shifts between the different fluid compartments in the brain. Aquaporin water channels have been recognized to have an important impact on the development of edematous swelling in the brain. Astrocytes, which are believed to induce or at least maintain the blood-brain barrier in the brain capillary endothelial cells, express the aquaporin isoform AQP4. Normally, AQP4 is highly concentrated in the glial membrane where astrocytes contact mesenchymal space, such as perivascular or brain superficial regions. Parenchymal membranes do not show any immunocytochemical AQP4-specific signal. We investigated the AQP4 expression in human glioblastoma and correlated it with the expression pattern of the extracellular heparan sulfate proteoglycan agrin and members of the dystrophin-dystroglycan complex. We found that AQP4 completely covered the surface of the glioma cells. -Dystroglycan was absent from glial membranes but retained in endothelial membranes. Utrophin and dystrophin remained restricted to the endfoot membrane in those cells in which AQP4 had been redistributed, whereas -syntrophin redistributed together with AQP4 across the entire cell surface. Since -dystroglycan operates as a binding protein for agrin, these observations support the suggestions that (1) AQP4 is tightly associated with the dystrophin-dystroglycan complex, and (2) agrin is necessary for the polarized distribution of AQP4 in the astrocyte. The results are discussed in connection with the fact that normally AQP4 is assembled in the so-called orthogonal arrays of particles (OAPs).The restriction of AQP4/OAPs to the endfoot membrane may be dependent on the presence of agrin, and this might be essentially connected to the ability of astrocytes to maintain the integrity of the blood-brain barrier.     

Effects of propofol on neuronal apoptosis and aquaporin-4 expression in a rat model of traumatic brain injury

BACKGROUND： Several studies have demonstrated that propofol exhibits protective effects in the central nervous system. OBJECTIVE： To observe the effects of propofol on neuronal apoptosis and aquaporin-4 （AQP-4） expression in a rat model of traumatic brain injury and to further investigate the mechanisms of action. DESIGN, TIME AND SETTING： The present neuronal, pathomorphological experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical College between April 2007 and March 2008. MATERIALS： Traumatic brain injury was induced by free falling objects in 150 healthy, male, Wistar rats. Propofol was produced by AstraZeneca, China. Rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemistry kit, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling （TUNEL） kit were purchased from Wuhan Boster Bioengineering Co., Ltd., China. METHODS： All 150 rats were randomly and evenly divided into lesion-only and propofol-treated groups. One hour after traumatic brain injury, propofol-treated animals received 1% propofol （10 mg/kg） through the caudal vein, followed by a sustained perfusion of 30 mg/kg propofol per hour for 2 hours, while the lesion-only group received equal volumes of physiological saline in parallel. MAIN OUTCOME MEASURES： At 6, 12, 24, 48, and 72 hours after traumatic brain injury, morphological changes in the peritraumatic and adjacent brain areas were analyzed in all rats by hematoxylin-eosin （HE） staining. In addition, cellular apoptosis was detected by TUNEL assay and the number of AQP-4-positive cells was determined by immunohistochemistry techniques. Brain water content was calculated as the ratio of dry to wet tissue weight. RESULTS： HE staining results demonstrated that, in the lesion-only group, the peritraumatic area exhibited neuronal and glial cell necrosis and disintegration. The adjacent area displayed swollen neuronal perikarya and vascular endothelial ceils, cellular edema, and a small amount of     

Propofol administration improves neurological function associated with inhibition of pro-inflammatory cytokines in adult rats after traumatic brain injury

BackgroundNeurological deficits following traumatic brain injury (TBI) result in dramatic impacts on the survivors, but the effect of propofol and associated mechanism are waiting to be determined.MethodsAdult male Sprague–Dawley rats were randomly assigned into Sham, TBI, TBI + Intralipid and TBI + Propofol group. Modified Feeney method was adopted to generate TBI model from free hammer fall injury, and animals in TBI + Propofol group were immediately treated with propofol administration for 2 hours after TBI, rats after TBI without propofol treatment was used as injury control, intralipid as vehicle in propofol was injected in TBI + intralipid group. Then, neurological severity scores (NSS) were evaluated at 1, 3, 7 and 14 days. Moreover, the expressions of IL-1β, IL-6 and TNF-α mRNA and protein were examined using quantitative real time-polymerase chain reaction and Western blot, immunohistochemical staining was used to localize cytokines.ResultsThe NSS increased greatly in the rats induced by TBI, while propofol could effectively decreased NSS, confirming the neuroprotective effect of propofol. Moreover, the mRNA expressions of IL-1β, IL-6 and TNF-α, at 1, 3, 7 days after operation (dpo), were significantly augmented in the injured cortex, compared with sham one. But there was no difference between TBI and TBI + Intralipid group, but markedly decreased after propofol treatment. Additionally, the protein level of IL-1β, IL-6 and TNF-α in four groups determined by Western blot and immunohistochemistry showed the similar change with mRNA expression.ConclusionPropofol treatment could elicit a robust neuroprotective response, resulting in significant neurological function improvement for TBI rats, which was independent with intralipid. The underlying molecular mechanism may be partially associated with an inhibition of pro-inflammatory cytokines.     

Removal of aquaporin-4 from glial and ependymal membranes causes brain water accumulation

There is a constitutive production of water in brain. The efflux routes of this excess water remain to be identified. We used basal brain water content as a proxy for the capacity of water exit routes. Basal brain water content was increased in mice with a complete loss of aquaporin-4 (AQP4) water channels (global Aqp4^−/− mice), but not in mice with a selective removal of perivascular AQP4 or in a novel mouse line with a selective deletion of ependymal AQP4 (Foxj1-Cre:Aqp4^flox/flox mice). Unique for the global Aqp4^−/− mice is the loss of the AQP4 pool subjacent to the pial membrane. Our data suggest that water accumulates in brain when subpial AQP4 is missing, pointing to a critical role of this pool of water channels in brain water exit.     

Induction of pro-inflammatory cytokine mRNAs in the brain after peripheral injection of subseptic doses of lipopolysaccharide in the rat.

Although it is generally accepted that pro-inflammatory cytokines produced by cells of the central nervous system play important roles in the communication between the central nervous system and the immune system during sepsis, it is not clear whether these cytokines are produced in the brain under subseptic conditions. In this study, we used in situ hybridization to examine the mRNA expression of the pro-inflammatory cytokines IL-1beta and TNFalpha in the brains of rats 2 and 12 h after they were challenged by peripheral injections of lipopolysaccharide (LPS) ranging from 0.01 to 1000 microg/kg. Unlike septic doses of LPS (> 500 microg/kg), which induce global expression of pro-inflammatory cytokines in the brain, subseptic doses of LPS (0.01-10 microg/kg) induced IL-1beta and TNFalpha mRNA expression only in the choroid plexus, the circumventricular organs, and meninges. The expression of the cytokine-responsive immediate early gene I kappaB alpha was induced in the brain after doses of LPS as low as 0.1 microg/kg. I kappaB alpha mRNA expression was confined to sites where IL-1beta and TNFalpha were expressed. These results indicate that the induction and action of pro-inflammatory cytokines during subseptic infection occur at the blood-brain barrier and at circumventricular organs, which may be sites for elaboration of signal molecules that communicate peripheral immune status to the brain.     

高压氧对颅脑损伤大鼠脑组织水通道蛋白-4表达的影响

目的 通过研究高压氧治疗对颅脑损伤大鼠脑组织水通道蛋白-4(AQP-4)表达与脑水肿的影响,探讨高压氧治疗颅脑损伤的可能作用机制. 方法 将雄性SD大鼠按随机数字表法分成3组,即假手术组、外伤对照组和高压氧治疗组,其中外伤对照组和高压氧治疗组均分为致伤后12h、1 d、3d及5 d4组.采用自由落体法制作颅脑损伤模型.干湿比重法测定脑组织的含水量,免疫组化法测定AQP-4的表达.结果 高压氧治疗组脑组织含水量较外伤对照组明显减少,但较假手术组增多,比较差异均有统计学意义(P<0.05).AQP-4在各组大鼠脑组织中均有表达,假手术组呈低表达;颅脑损伤后12 h伤灶周围水肿区星形胶质细胞足突AOP-4表达开始增高,1 d达到高峰,3 d后降低;高压氧组治疗大鼠各时间点伤灶周围AQP-4的表达与外伤对照组相比明显减低,差异具有统计学意义(P<0.05).结论 高压氧治疗可能通过AQP-4表达的减少来减轻脑水肿的发生,从而保护病灶周围脑组织.     

Increased pro-inflammatory cytokine gene expression in peripheral blood mononuclear cells of patients with polyneuropathies

Background

Distinct cytokine expression patterns have been reported in biomaterial of patients with polyneuropathies (PNP). We investigated gene expression profiles of pro- and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMC) of patients with neuropathies of different etiologies.

Methods

We prospectively studied 97 patients with neuropathies and compared data between diagnostic subgroups and healthy controls. Gene expression of a panel of pro- and anti-inflammatory cytokines was analyzed (interleukin-1 [IL-1], IL-2, IL-6, IL-8, tumor necrosis factor alpha [TNF], IL-4, and IL-10) in PBMC samples. Furthermore, protein levels of IL-6, IL-8, and TNF were measured in supernatant of PBMC stimulated with lipopolysaccharide (LPS).

Results

PNP were associated with higher PBMC gene expression of IL-1 (p < 0.05), IL-2 (p < 0.05), IL-8 (p < 0.001), and TNF (p < 0.01) compared to healthy controls. Inflammatory neuropathies were associated with higher gene expression of IL-8 (p < 0.001) and TNF (p < 0.05) and lower gene expression of IL-10 (p < 0.05) compared to healthy controls. More pro-inflammatory cytokines were elevated in painful neuropathy (IL-1, IL-2 [p < 0.05], IL-8 [p < 0.001] and TNF [p < 0.05]) than in painless neuropathy (IL-8 [p < 0.01] and TNF [p < 0.01]) compared to healthy controls, while IL-10 expression was higher in treatment naïve patients with painless neuropathy compared to patients with painful neuropathy (p < 0.05). Disease duration positively correlated with IL-6 gene expression (p < 0.01). Supernatant protein levels of IL-6, IL-8, and TNF did not differ between groups.

Conclusion

Systemic gene expression of pro-inflammatory cytokines is increased in patients with neuropathies and may be influenced by the presence of neuropathic pain.